Polyketide antibiotics include many of the most commonly used antibiotics in medicine today such as erythromycin, rapamycin and avermectin. In Part 1, Dr. Khosla describes the modular enzymes that synthesize these antibiotics, with each module adding to a growing polyketide chain in an assembly-line manner. Using the enzyme 6-deoxyerythronolide B synthase (DEBS) as an example, he walks us through the multiple steps from simple precusor to complex product. Khosla also explains that a vast improvement in DNA sequencing in the past decade has led to the identification of many gene clusters encoding polyketide assembly lines. The products of these assembly lines are not known, however, suggesting that new and possibly useful antibiotics are yet to be discovered.
Many of the polyketide assembly line enzymes are found in unusual bacteria or other organisms. In Part 2, Khosla explains that he and his colleagues have expressed the DNA encoding DEBS in E.coli. When the appropriate precursors and energy sources are added to these modified E.coli, they efficiently produce 6-deoxyerythronolide B. This system facilitates the study of each step of the DEBS assembly line both in vitro, using proteins purified from the system, and in vivo. Using these tools, Khosla and colleagues are able to probe the stereospecificity and side chain specificity of the assembly line. They are also learning how to efficiently rewire individual modules to produce novel polyketide products.
In Part 3, Khosla asks how nature ensures that the product of one module in a polyketide synthesis pathway is passed on to the correct, subsequent module in the pathway? How is it that reactive intermediates are directed vectorially along the assembly line? Khosla explains that vectorial specificity is due to the protein-protein interactions of the faces of the different protein modules in the pathways.
00:00:08.25 This lecture,
00:00:10.13 or this trilogy of lectures,
00:00:13.12 is on the assembly-line biosynthesis
00:00:16.19 of a class of antibiotics
00:00:19.06 called the polyketide antibiotics.
00:00:22.28 Before I start,
00:00:25.08 I would like to share with you
00:00:27.11 the motivation
00:00:29.06 for recording a set of lectures
00:00:31.24 that I did in a similar setting
00:00:35.21 more than a decade ago.
00:00:39.23 There's three things that have changed.
00:00:42.26 The first one is that
00:00:45.11 the field of polyketide antibiotic biosynthesis
00:00:48.24 has moved forward quite a bit
00:00:51.14 over the past decade
00:00:53.10 and I thought it would be helpful for you
00:00:55.16 to know what has changed.
00:00:57.21 The second one is that the world of science,
00:01:00.15 and biochemistry in particular,
00:01:02.14 in which this topic sits,
00:01:04.29 has moved significantly too,
00:01:08.15 and so it would be helpful for you
00:01:10.29 to place what is new and important in this field
00:01:15.11 in the broader context of biochemistry.
00:01:18.19 And the third one, of course,
00:01:20.12 is I've gotten ten years older.
00:01:23.03 And while the latter generally
00:01:25.20 leads to more gray hair,
00:01:27.16 in my case considerably more
00:01:29.27 than what was there in the first version that I recorded,
00:01:34.08 it also gives somebody like myself,
00:01:37.04 who's been working on this problem
00:01:39.12 for more than two decades,
00:01:41.23 a chance to think about
00:01:44.01 where this field is going,
00:01:45.17 and I hope some of the things I share with you today
00:01:48.10 might give, especially the young people in the audience,
00:01:53.02 a chance to think about
00:01:55.09 where the field could be ten years from now
00:01:57.16 thanks to your own efforts.
00:01:59.19 Okay, so with that as a backdrop,
00:02:02.23 let me start with what you're looking at:
00:02:05.24 this picture of an automobile assembly line.
00:02:10.08 You all instantly recognize
00:02:13.10 what you're looking at in this picture.
00:02:16.19 You're looking at Henry Ford's contribution
00:02:20.07 to modern society.
00:02:22.21 And what you're seeing, more specifically,
00:02:25.09 is an assembly line
00:02:27.20 that builds cars
00:02:30.04 on a series of way stations
00:02:33.22 where at each way station
00:02:37.05 there are a set of catalysts,
00:02:39.01 human and mechanical,
00:02:42.03 that perform exquisite tasks
00:02:45.08 with a lot of sophistication,
00:02:48.03 but more or less
00:02:51.03 in a manner that is
00:02:54.03 oblivious about what is happening
00:02:56.23 upstream or downstream
00:02:59.24 of their way station.
00:03:02.14 And the genius of Henry Ford
00:03:05.04 lies in the modularity of this device.
00:03:09.28 So, the same assembly line
00:03:12.08 that builds a Ford Escort,
00:03:15.00 by changing a few things at the way stations
00:03:18.27 -- you could change some of the catalysts
00:03:21.03 that do certain operations
00:03:24.09 at a way station,
00:03:26.00 or you could change the inputs
00:03:28.26 at different points in the way station --
00:03:31.04 by those relatively simple and modular changes,
00:03:35.00 you can change the output,
00:03:37.04 which in one case could be a Fort Escort,
00:03:41.06 in another case might be a Lincoln Town Car
00:03:44.25 or your favorite automobile.
00:03:49.09 Now, nature has come up with a very similar strategy
00:03:53.05 to make a class of antibiotics
00:03:56.15 called the polyketide antibiotics,
00:03:59.28 and what I show you in this simple cartoon
00:04:03.10 is a prototypical example
00:04:06.25 of an assembly line
00:04:09.11 that's made up of a bunch of enzymes
00:04:11.13 and is responsible for making
00:04:14.10 a key intermediate
00:04:16.25 in the biosynthesis of the well-known antibiotic
00:04:22.01 This intermediate is called
00:04:23.24 6-Deoxyerythronolide B
00:04:27.08 and the assembly
00:04:28.24 that makes 6-Deoxyerythronolide B
00:04:31.14 is called the 6-Deoxyerythronolide B Synthase,
00:04:35.27 or DEBS for short.
00:04:38.25 And DEBS is made up
00:04:42.10 of three very large proteins.
00:04:46.20 You're seeing those three proteins
00:04:49.02 in cartoon forms on this slide.
00:04:53.15 Each protein,
00:04:55.11 you see the first protein made up of two modules
00:04:59.07 -- Modules 1 and 2 --
00:05:01.27 and an upstream bit
00:05:03.23 that we call the loading domain,
00:05:05.21 or LD for short.
00:05:08.00 And this protein is a homodimer.
00:05:11.02 You then have a second protein
00:05:13.29 that is also a homodimer
00:05:15.27 and is made up of two more modules of catalysts,
00:05:19.22 and then a final protein
00:05:21.26 that is made up of two additional modules
00:05:25.01 and another catalyst
00:05:27.11 called the TE, or thioesterase for short.
00:05:31.00 So this alpha2-beta2-gamma2 hexamer
00:05:36.17 that makes up this assembly line called DEBS,
00:05:41.23 has a molecular mass
00:05:45.07 of a little bit over 2 million daltons.
00:05:48.24 For those of you who don't know what 2 million daltons
00:05:51.24 buys you in biochemistry,
00:05:54.10 that's about the size of a ribosome.
00:05:57.08 And so, as a point of curiosity,
00:06:00.13 you may wish to acknowledge
00:06:03.01 that it takes nature a 2 million dalton machine
00:06:07.04 to make an antibiotic
00:06:09.04 whose job it is to gum up
00:06:11.03 the other 2 million dalton machine.
00:06:14.10 The rest of my lecture
00:06:15.29 is gonna be focused
00:06:18.10 on this assembly line DEBS.
00:06:21.05 Now, DEBS is an assembly line
00:06:24.25 that uses a bunch of precursors
00:06:28.16 that are available in metabolism.
00:06:31.15 You all are familiar with precursors
00:06:34.08 such as acetyl coenzyme A (acetyl-CoA)
00:06:38.15 or malonyl coenzyme A.
00:06:42.01 What you're seeing in this slide
00:06:44.14 are subtle variants of acetyl coenzyme A
00:06:49.00 and malonyl coenzyme A.
00:06:51.13 You're seeing a precursor
00:06:53.22 called propionyl coenzyme A
00:06:55.27 on the far left of this slide,
00:06:58.24 and the central precursor
00:07:00.27 that feeds into each of the six modules
00:07:05.15 of the assembly line
00:07:07.12 is a variant of malonyl coenzyme A
00:07:09.27 called methylmalonyl coenzyme A.
00:07:12.16 And so nature crafts this product,
00:07:16.23 6-Deoxyerythronolide B,
00:07:19.29 out of one equivalent of propionyl coenzyme A,
00:07:25.07 six equivalents of methylmalonyl coenzyme A,
00:07:29.28 and six equivalents of NADPH,
00:07:33.05 which you all know
00:07:34.29 is a reducing equivalent in biology.
00:07:39.16 And the way these precursors
00:07:42.08 come together to make
00:07:45.10 this molecule 6-Deoxyerythronolide B
00:07:49.09 that you're seeing to the far right of this assembly line
00:07:53.14 is by a mechanism
00:07:56.01 where you have propionyl coenzyme A
00:07:59.17 that starts the assembly line,
00:08:01.26 that primes the assembly line,
00:08:04.14 and through incremental addition of precursors,
00:08:08.11 you have at each module
00:08:11.22 on the assembly line,
00:08:13.08 you have further elaboration
00:08:15.21 of the precursor
00:08:18.04 to give you a highly complex product
00:08:22.14 at the end
00:08:24.15 called 6-Deoxyerythronolide B.
00:08:27.08 And this assembly line was discovered independently
00:08:30.29 by two research groups:
00:08:32.26 one at the University of Cambridge,
00:08:35.29 and another at Abbott Laboratories,
00:08:39.04 both who were working on this problem
00:08:42.01 about 25 years ago.
00:08:45.19 Now, just like Erythromycin,
00:08:48.18 there are a number of other complex antibiotics
00:08:52.24 that are made by this
00:08:58.02 assembly line strategy,
00:09:00.25 and you're seeing some of the who's who
00:09:03.12 among antibiotics
00:09:05.10 on this slide,
00:09:06.28 each of which is made
00:09:08.21 by a biosynthetic assembly line
00:09:10.27 that's similar, very similar,
00:09:12.20 to the kind that's used to make
00:09:15.02 6-Deoxyerythronolide B.
00:09:18.05 So, here's the outline
00:09:20.09 of what I have to say to you today.
00:09:24.02 I will first...
00:09:26.04 this module is going to focus
00:09:29.02 on three general overview topics
00:09:32.06 that I expect should be accessible
00:09:36.10 to anybody who has had,
00:09:38.12 or is concurrently taking,
00:09:40.28 a basic course in biochemistry.
00:09:43.25 I am going to tell you about
00:09:46.10 the evolutionary biology
00:09:49.00 of these assembly lines.
00:09:51.13 I will then talk a little bit about
00:09:53.15 the chemistry that happens
00:09:55.20 on the DEBS assembly line,
00:09:58.00 and then I'll give you an overview of
00:10:00.06 what this assembly line actually looks like
00:10:02.19 so you can put everything else in context.
00:10:06.21 In subsequently modules,
00:10:08.18 we'll talk about the tools the field uses
00:10:12.02 to study these assembly line enzymes
00:10:14.25 and then some properties
00:10:17.04 of these assembly lines
00:10:19.03 that represent cutting-edge topics
00:10:21.06 in modern research.
00:10:23.20 So let's start with the biology.
00:10:26.03 Back when we started working
00:10:28.01 on this assembly line,
00:10:30.10 which is way out here
00:10:32.13 perhaps even before then,
00:10:34.18 there was only one assembly line
00:10:36.27 that was known: DEBS.
00:10:39.26 And so you either worked on DEBS
00:10:41.23 or you did something else.
00:10:44.08 As you can see in this graph,
00:10:47.28 the world has changed significantly
00:10:50.10 over the past 20 years.
00:10:52.23 It had changed some,
00:10:54.09 but not a whole lot,
00:10:56.09 around the time I recorded
00:10:58.16 the earlier version of these lectures.
00:11:01.27 There were maybe a few tens
00:11:04.02 of these assembly lines
00:11:05.23 that had been painstakingly cloned
00:11:07.20 and sequenced
00:11:09.19 over the first 10 or 15 years on this slide,
00:11:14.00 and then something happened
00:11:16.02 around the mid-2000s.
00:11:18.12 As I'm sure most of you recognize,
00:11:20.18 that's around the time
00:11:22.07 it became relatively easy
00:11:24.01 to sequence genomes,
00:11:26.13 in particular bacterial genomes,
00:11:29.00 and since then the field
00:11:32.13 has exploded
00:11:34.10 in terms of the number of assembly lines
00:11:37.07 that are known to us
00:11:39.18 through sequence identity.
00:11:42.00 So, as of last summer,
00:11:44.22 there were close to 1000
00:11:47.12 distinct polyketide assembly lines
00:11:50.22 that had been cloned and sequenced,
00:11:52.28 and whose sequence had been deposited
00:11:55.10 in the database.
00:11:57.12 Now, what's important to note
00:11:59.09 about this slide
00:12:01.24 is a vast majority
00:12:04.07 of the assembly lines
00:12:05.27 whose sequence is available today
00:12:08.15 are what we call
00:12:10.09 orphan assembly lines.
00:12:12.09 Nobody really knows
00:12:14.04 what these assembly lines are doing in nature.
00:12:16.20 We just know their sequence.
00:12:18.18 And so we know they must be doing
00:12:20.28 something in nature,
00:12:22.12 or they probably are doing something,
00:12:24.26 but a vast majority,
00:12:26.23 about 80% of these assembly lines,
00:12:29.29 are begging for insights.
00:12:33.13 Here is an evolutionary tree
00:12:35.20 -- some of you may recognize it
00:12:37.13 as looking like a dendrogram --
00:12:39.21 of about 50 of the known
00:12:44.00 polyketide assembly lines
00:12:45.22 that have been sequenced to date.
00:12:48.07 And I don't expect you to read this slide
00:12:50.24 in detail,
00:12:53.10 but for those of you who have
00:12:55.26 heard about polyketide antibiotics,
00:12:58.06 to the right of this slide
00:12:59.26 what you're seeing are names
00:13:01.27 like Macrolides or Macrolide antibiotics,
00:13:06.05 FKBP-binding antibiotics
00:13:08.10 like FK-506 and Rapamycin,
00:13:11.10 Polyether antibiotics
00:13:13.09 that are frequently used in veterinary medicine.
00:13:16.09 These are...
00:13:18.00 Ansamycins, which include Rifamycin,
00:13:21.00 the front-line antibiotic to treat tuberculosis.
00:13:25.09 These are all antibiotics
00:13:28.02 that represent the who's who of infectious diseases,
00:13:32.09 cancer chemotherapy,
00:13:34.03 and related disease states.
00:13:37.01 These are a few examples
00:13:38.22 of polyketide assembly lines
00:13:40.21 whose sequence we know today.
00:13:44.07 But these are only a small fraction
00:13:47.10 of the polyketide assembly lines
00:13:51.28 whose existence we know of today.
00:13:54.14 So, what you're seeing on the far left
00:13:57.02 of this slide
00:13:59.23 is another family tree,
00:14:02.06 another dendrogram,
00:14:04.09 that I'm almost certain nobody can read
00:14:07.00 and I don't expect you to.
00:14:09.07 The point I want to leave you with
00:14:11.17 is that this vast pool
00:14:14.05 of orphan assembly lines
00:14:16.19 represents a really interesting starting point
00:14:20.04 for a field that's looking forward,
00:14:23.27 because the known polyketide assembly lines
00:14:28.01 today represent only small fractions
00:14:32.16 of this overall dendrogram.
00:14:35.11 There are large swaths of this family tree
00:14:39.05 where we know nothing about
00:14:41.05 what these polyketide synthases are doing.
00:14:44.27 And here's just one interesting factoid:
00:14:47.16 a lot of people who look at this field think,
00:14:50.25 "Oh, these are antibiotics biosynthetic enzymes,
00:14:53.11 they exist in bacteria."
00:14:55.20 That's true; many, perhaps most of these
00:14:59.05 antibiotic assembly lines exist in bacteria.
00:15:02.22 But this arrow down toward the bottom
00:15:06.11 of this slide
00:15:08.08 points to a small clade
00:15:10.11 in this very large
00:15:13.13 collection of orphans
00:15:16.00 that actually is encoded
00:15:18.12 by a bunch of worms.
00:15:20.19 And if you look a little bit further
00:15:22.22 at some of these assembly lines,
00:15:25.01 they seem to be making
00:15:27.06 some fairly complicated antibiotics.
00:15:29.14 Again, I want to emphasize
00:15:31.00 we don't know what these assembly lines
00:15:32.29 are making for certain,
00:15:34.23 but we can be reasonably confident
00:15:36.25 that these assembly lines
00:15:38.17 are making something very complex,
00:15:40.18 and they probably are doing so
00:15:42.16 for the benefit of the worms,
00:15:44.29 and we don't understand
00:15:48.01 what or how.
00:15:50.17 So, this field of polyketide biosynthesis,
00:15:54.22 one of the major things
00:15:56.19 that has happened is,
00:15:59.00 back when I recorded this in the past,
00:16:03.13 for any one of these assembly lines,
00:16:06.10 if you wanted the genetic information,
00:16:09.04 the blueprint for these assembly lines,
00:16:11.28 that would be close to an entire PhD thesis.
00:16:16.02 Today, you can get
00:16:19.01 thousands of these assembly lines
00:16:21.04 essentially for the price of free.
00:16:25.11 These exist in the database
00:16:27.05 and you can do whatever you want.
00:16:29.12 And so there's two major challenges
00:16:31.20 for the field going forward,
00:16:33.14 starting with this embarrassment of riches.
00:16:37.05 The first one is to develop the knowledge
00:16:40.07 that can help us decode
00:16:42.19 what these assembly lines
00:16:44.06 are doing in nature,
00:16:46.07 because that insight might let us
00:16:49.02 exploit the products of these orphan assembly lines
00:16:52.11 for interesting medical
00:16:54.07 and/or other applications.
00:16:57.04 The second thing
00:16:58.25 that one could hope the field can deliver
00:17:01.02 over the coming decade
00:17:03.03 is the insights
00:17:05.19 that might allow us to start
00:17:08.00 with these 1000 or 2000 assembly lines
00:17:11.05 and scramble them in ways
00:17:13.24 to make molecules that nature
00:17:17.02 probably didn't bother trying out,
00:17:19.05 or maybe nature tried out
00:17:20.18 but didn't find much use for,
00:17:22.21 but humanity could find use for.
00:17:25.29 And that represents another
00:17:27.17 important goal in the field.
00:17:30.28 Okay, so with that as a biological background,
00:17:33.15 let's switch to the chemistry
00:17:35.10 that happens on one of these
00:17:37.10 polyketide assembly lines,
00:17:39.07 namely DEBS.
00:17:40.22 So, I introduced you to this assembly line,
00:17:45.04 that makes this intricate molecule
00:17:47.06 to the right
00:17:48.21 called 6-Deoxyerythronolide B.
00:17:50.24 Perhaps the best way for me
00:17:53.10 to give you a sense of the actual
00:17:55.02 enzymatic chemistry
00:17:56.24 that's happening on this assembly line
00:17:59.03 is by zeroing in
00:18:01.13 on one of those modules,
00:18:03.18 and I've boxed Module 3
00:18:06.09 for illustration purposes,
00:18:08.24 and let's take a close look
00:18:10.29 at what's happening in Module 3,
00:18:13.21 as well as its two interfaces
00:18:16.07 with its neighboring modules,
00:18:18.07 Module 2 and Module 4, respectively.
00:18:21.23 Because if we can understand
00:18:23.22 what Module 3 does
00:18:25.28 in the overall biosynthetic process,
00:18:29.03 how it talks to Module 2
00:18:31.25 and how it hands off its produc
00:18:34.28 t to Module 4,
00:18:36.20 then the rest of this pathway
00:18:39.05 becomes relatively straightforward
00:18:41.06 to understand.
00:18:43.08 So, in order to explain to you
00:18:45.16 what Module 3 does,
00:18:48.09 we need to go into
00:18:50.08 a higher level of granularity,
00:18:52.16 and this is where I have introduced
00:18:54.16 a few acronyms in the slide
00:18:57.10 for those of you who are still paying attention,
00:19:00.06 you've started to see,
00:19:01.25 in what I had earlier on called
00:19:04.14 Modules 2, 3, and 4,
00:19:06.11 the appearance of some acronyms
00:19:09.07 'KS', 'AT', 'KR', 'ACP', and so on.
00:19:16.00 For the initiated,
00:19:18.14 these are the quintessential
00:19:22.05 catalytic domains
00:19:24.04 that one finds in all of these
00:19:26.03 polyketide assembly lines.
00:19:28.12 For the uninitiated in you,
00:19:30.18 in the lower-left corner of this slide,
00:19:33.13 and in all subsequent slides
00:19:35.08 where I use these kinds of acronyms,
00:19:38.11 I'll keep a key
00:19:40.02 so that you can reference quickly
00:19:42.18 what kind of enzyme or protein
00:19:45.09 I'm talking about,
00:19:46.23 and so when you see
00:19:48.15 the letters KS in one of these modules,
00:19:52.15 you know I am talking about
00:19:54.25 a ketosynthase,
00:19:56.25 and I'll introduce you to the enzymology
00:19:59.09 of a ketosynthase
00:20:01.06 in a moment.
00:20:02.20 Similarly, when you see the letters
00:20:04.21 AT, I'm talking about an acyltransferase.
00:20:08.09 When you see the letters
00:20:10.00 KR, I'm talking about a ketoreductase,
00:20:14.03 and so on and so forth.
00:20:17.03 So in order to understand
00:20:18.29 what Module 3 does,
00:20:21.09 what we're gonna do
00:20:22.20 is we're gonna peel away
00:20:24.24 everything else in the assembly line
00:20:26.15 except for Module 3,
00:20:29.28 the ACP, or the acyl carrier protein
00:20:33.24 from the upstream module,
00:20:35.20 because that's the domain of the upstream Module 2
00:20:40.05 that donates the polyketide chain
00:20:42.12 into Module 3,
00:20:44.15 and the KS, or ketosynthase
00:20:47.19 from Module 4, because that is the recipient
00:20:50.20 of the product of Module 3.
00:20:53.27 And now we can look
00:20:56.03 at the catalytic cycle
00:20:58.16 associated with Module 3.
00:21:00.26 So we're gonna start
00:21:02.22 from the state of this module
00:21:05.12 that is shown at 10 o'clock
00:21:08.18 on this catalytic cycle,
00:21:11.02 where you see that the Module 3 itself
00:21:14.22 is empty;
00:21:16.06 it has no precursors bound to it.
00:21:19.02 There is a substrate
00:21:22.03 that is bound to the upstream
00:21:24.22 acyl carrier protein,
00:21:26.17 which is ready to come into Module 3,
00:21:29.04 and Module 3,
00:21:31.05 from its previous catalytic cycle,
00:21:32.29 has donated its product
00:21:35.12 to the next module,
00:21:37.13 Module 4.
00:21:39.12 So, starting from that point,
00:21:41.15 the first chemical step that needs to occur
00:21:44.10 is a translocation event.
00:21:47.03 In this event,
00:21:48.26 the growing polyketide chain,
00:21:51.12 which was a triketide
00:21:54.06 on the acyl carrier protein
00:21:57.02 of the upstream module,
00:21:59.16 has been moved into
00:22:02.22 the Module 3
00:22:04.16 and is now bound
00:22:06.17 to the ketosynthase
00:22:08.12 through what you might recognize
00:22:10.06 as a thioester linkage.
00:22:13.04 So the substrate
00:22:15.05 was an acyl protein carrier-bound thioester,
00:22:18.18 and the product is also a thioester,
00:22:21.17 but now it's bound in the underbelly
00:22:24.27 of Module 3
00:22:26.25 at the ketosynthase active site.
00:22:29.27 That reaction, from here on out,
00:22:32.15 we're gonna talk about
00:22:34.06 as a translocation event,
00:22:36.04 because the chain has been translocated
00:22:38.20 from one module to the next.
00:22:42.13 The next event in the catalytic cycle
00:22:44.17 is an acyl transfer event.
00:22:47.24 This is when Module 3
00:22:50.04 makes a critical choice
00:22:52.03 about what precursor
00:22:54.02 it is gonna pick from the cell soup,
00:22:56.20 from the metabolic pool of precursors
00:22:59.14 that exists in the cell,
00:23:01.20 and bring it inside
00:23:03.19 so it can catalyze
00:23:05.22 the next set of operations,
00:23:08.02 and this event we're gonna call
00:23:09.29 acyl transfer.
00:23:12.03 It is catalyzed by the acyltransferase,
00:23:15.15 or AT for short,
00:23:17.21 and you'll see Module 3
00:23:19.12 has one of those domains in it.
00:23:22.08 And the acyltransferase of Module 3
00:23:25.17 has picked
00:23:27.16 a methylmalonyl extender unit
00:23:30.15 from a coenzyme A-bound precursor
00:23:34.12 and transferred that methylmalonyl extender unit
00:23:38.00 onto the ACP, or acyl carrier protein,
00:23:42.25 of that module.
00:23:45.15 So now,
00:23:47.15 we are at about 3pm
00:23:49.28 of this catalytic cycle.
00:23:52.06 You have an electrophilic,
00:23:54.10 growing polyketide chain
00:23:56.13 attached as a thioester
00:23:58.28 to the ketosynthase.
00:24:01.05 You have a nucleophile,
00:24:02.27 the methylmalonyl extender unit
00:24:05.16 that is attached to the acyl carrier protein,
00:24:08.29 and the stage is set up
00:24:11.03 for the next reaction,
00:24:13.06 which we're gonna call
00:24:15.05 the elongation reaction.
00:24:17.08 This is where
00:24:19.03 you see a carbon-carbon bond
00:24:21.25 having formed
00:24:23.24 between this 3-carbon unit
00:24:25.21 and the rest of the chain
00:24:27.21 that you see
00:24:29.14 that came from the upstream module.
00:24:32.11 So, this reaction
00:24:34.06 is the elongation step
00:24:37.09 and it is, for those of you who are considering
00:24:40.08 the overall thermodynamics of the process,
00:24:43.11 this is the key energy-giving step
00:24:47.00 in the process.
00:24:49.03 From a thermodynamic perspective,
00:24:50.24 it's this release of CO2 in the step
00:24:53.27 that drives this overall enzymatic process forward.
00:25:00.09 The next reaction,
00:25:02.09 that I call chain modification,
00:25:05.03 is a variable set of operations
00:25:07.13 that this module does.
00:25:09.29 In this particular case,
00:25:12.07 all that the enzyme has done
00:25:15.06 is it's catalyzed
00:25:17.07 a racemization of the C2 carbon
00:25:21.13 of the newly elongated polyketide chain.
00:25:25.22 So the chain elongation step
00:25:30.21 involved what is known as an
00:25:33.16 inversion of stereochemistry.
00:25:36.02 The (2S)-methylmalonyl extender unit
00:25:39.21 stereochemistry got inverted,
00:25:42.24 and you have the product that is formed.
00:25:45.26 And in the modification step
00:25:51.07 that second carbon in the growing polyketide chain
00:25:54.22 is scrambled
00:25:57.02 to give you a racemic center.
00:25:59.26 That step is catalyzed
00:26:02.08 by the enzyme that I have designated in this module
00:26:05.14 as a KR0.
00:26:08.11 Now, for those of you who are
00:26:10.04 paying attention to the key I have
00:26:13.01 in the lower-left corner,
00:26:15.04 you're wondering
00:26:16.24 why did I call a racemase
00:26:19.06 a ketoreductase.
00:26:21.09 That will become apparent to you
00:26:23.08 as we go further in this lecture.
00:26:27.15 Once the modification occurs,
00:26:29.25 now you have both different flavors of stereochemistry available
00:26:36.14 and the chain is ready to move
00:26:38.17 into the next module,
00:26:40.20 which selective chooses
00:26:43.01 only one of those two diastereomers
00:26:46.16 to process further,
00:26:48.10 well the other one can be racemized again
00:26:51.12 by that ketoreductase-like racemase
00:26:54.14 to give you additional precursors
00:26:57.01 that can be moved forward.
00:26:59.10 And so the take-home message from this slide
00:27:02.28 is that a catalytic cycle
00:27:05.20 of a polyketide assembly line module
00:27:08.25 comprises of
00:27:11.29 two translocation events,
00:27:13.26 one from the upstream module,
00:27:15.16 one to the downstream module,
00:27:18.06 an acyl transfer that picks a building block,
00:27:21.19 a chain elongation event,
00:27:24.09 and one or more modification events
00:27:27.09 that leads to diversity generation
00:27:30.07 on the growing polyketide chain.
00:27:33.05 And now that you understand
00:27:35.01 what Module 3 does,
00:27:37.17 it becomes relatively easy for you
00:27:39.18 to see how each of the six modules
00:27:43.00 of the 6-Deoxyerythronolide B Synthase
00:27:47.00 perform a set of catalytic operations,
00:27:51.02 in each case
00:27:52.22 on a methylmalonyl extender unit,
00:27:55.06 and a unique incoming polyketide chain,
00:27:58.15 to give you the product
00:28:00.19 that comes out of this assembly line.
00:28:04.02 Okay, so now that you understand the chemistry
00:28:07.08 of this assembly line,
00:28:09.07 let's talk a little bit about the structure.
00:28:11.28 Now, clearly, I'm showing you...
00:28:14.14 you must recognize that even though I show you
00:28:17.02 this assembly line
00:28:18.17 in cartoon form as I showed you in this slide,
00:28:21.13 this is not what the system looks like in nature,
00:28:24.00 and many of you are probably already asking this question:
00:28:26.29 what does this assembly line
00:28:29.15 look like in three dimensions?
00:28:32.05 So, this is what we know today
00:28:35.12 about the 6-Deoxyerythronolide B [synthase].
00:28:40.00 So on the top of this slide
00:28:41.19 I show you that same assembly line
00:28:43.23 that I showed you earlier on,
00:28:46.01 with all those active sites
00:28:48.06 labeled the same way,
00:28:50.15 but now what I have highlighted
00:28:53.21 in green are the portions of the assembly line
00:28:58.13 whose atomic structures have been solved,
00:29:01.22 primarily by X-ray crystallography,
00:29:05.04 but also using NMR.
00:29:09.07 What you can see over here is,
00:29:11.22 we know today the atomic structures
00:29:14.10 of about a quarter
00:29:16.11 to a third of this overall assembly line.
00:29:19.17 These structures of the different pieces
00:29:22.11 that I've highlighted in green-blue
00:29:25.11 have been solved
00:29:28.16 by first extracting these pieces
00:29:30.18 out of the assembly line
00:29:33.08 and then solving the structures
00:29:36.22 of these pieces.
00:29:38.29 What is important to recognize is that
00:29:42.19 the DEBS assembly line
00:29:45.12 has a very strong
00:29:47.21 repetitive characteristic.
00:29:50.08 So, different active sites
00:29:52.14 occur again and again
00:29:54.12 in the assembly line.
00:29:56.07 What you will see is
00:29:58.03 there is a ketosynthase, KS,
00:30:00.01 that is associated with each of the six core modules
00:30:04.01 of the assembly line,
00:30:05.29 as is an acyltransferase, or AT,
00:30:08.20 or an ACP.
00:30:10.18 So, what you have are
00:30:13.01 domains that have homologues,
00:30:15.19 and these are very homologous domains,
00:30:17.28 so any two of the ketosynthases
00:30:20.16 in the 6-Deoxyerythronolide B Synthase
00:30:23.20 have upwards of 50% identity.
00:30:26.17 And through this divide and conquer approach,
00:30:30.17 one can therefore derive
00:30:33.05 a fairly good insight
00:30:35.10 into what the atomic structures
00:30:37.14 of any of the individual domains
00:30:40.15 within the assembly line are.
00:30:42.16 So, we have atomic structures
00:30:44.23 of at least one prototypical domain
00:30:48.01 of all of these active sites
00:30:51.22 that comprise the DEBS enzymatic assembly line.
00:30:55.25 So, that information...
00:30:57.19 starting from that information,
00:31:00.02 one can now start to build models
00:31:02.24 for what an actual catalytic module
00:31:05.29 might look like.
00:31:08.26 This cartoon that I show you here
00:31:12.12 is our best guess...
00:31:15.08 so at the bottom of this cartoon
00:31:17.05 you're seeing, in color,
00:31:19.19 the DNA arrangement of the domains
00:31:23.29 that make up one of these modules
00:31:26.25 that contains a ketosynthase,
00:31:28.24 an acyltransferase,
00:31:31.01 a ketoreductase,
00:31:32.14 acyl carrier protein,
00:31:34.02 and so on.
00:31:36.00 And at the same time
00:31:37.22 what you're seeing on the top
00:31:39.19 is a model
00:31:41.21 for what this module might look like
00:31:44.12 in three dimensions.
00:31:46.15 Some aspects that you're seeing in this model,
00:31:49.08 in the structural model,
00:31:52.08 are hard facts,
00:31:53.27 because we have actual X-ray crystallographic structures
00:31:56.29 or NMR structures
00:31:58.26 of the pieces,
00:32:00.17 but the relative orientations of these...
00:32:03.05 so for example,
00:32:04.27 the relative orientation of the pale blue part on the top
00:32:08.10 and the lower butterfly-like structure
00:32:12.13 is somewhat speculative,
00:32:15.05 and it's been derived
00:32:17.03 primarily through models
00:32:19.19 that compare this polyketide synthase
00:32:22.26 with the vertebrate fatty acid synthase
00:32:25.15 that is a homologue of this module,
00:32:27.24 and whose structure has been solved.
00:32:31.16 Now, how might one get additional data
00:32:34.20 on what these modules might look like?
00:32:37.12 This is where one uses
00:32:39.16 lower resolution methods,
00:32:41.20 in our case over here
00:32:43.22 we've used SAXS,
00:32:46.05 or small-angle X-ray scattering.
00:32:48.27 The graph that I show you to the left
00:32:52.08 is a typical plot
00:32:54.23 that one gets of scattering intensity
00:32:57.07 against scattering angle,
00:32:59.13 and from that data
00:33:01.08 one can get information
00:33:03.09 about the size and shape
00:33:06.05 of the protein
00:33:08.05 that has been subjected to SAXS analysis.
00:33:11.10 And from that information
00:33:13.03 about size and shape,
00:33:14.28 one can derive
00:33:16.28 lower-resolution, but still useful models
00:33:19.29 for what the module might look.
00:33:22.11 And what you're seeing in this slide
00:33:24.05 suggests that the model I showed you
00:33:26.17 in the earlier slide,
00:33:28.09 which was derived from homology
00:33:29.28 with the fatty acid synthase,
00:33:31.19 isn't too far off-base.
00:33:34.13 You can use SAXS also
00:33:37.27 to look at larger pieces of the assembly line.
00:33:41.06 So, here we're looking at the SAXS data,
00:33:47.15 scattering data
00:33:49.02 derived from a very large
00:33:51.10 two-module protein
00:33:54.09 that has a homodimeric molecular mass
00:33:56.23 on the order of 650 kiloDaltons.
00:34:00.05 Again, the key take-home message
00:34:02.21 that you wanna take from this slide is
00:34:05.10 that there is a very defined three-dimensional architecture
00:34:09.05 that one can predict
00:34:11.13 for these assembly lines,
00:34:13.21 or in this case
00:34:15.05 two adjacent modules of the assembly line.
00:34:17.29 And if you put this kind of model building together,
00:34:21.14 what you can get is insight into,
00:34:24.29 or at least a working model
00:34:29.09 for what the overall assembly line
00:34:31.12 might look like.
00:34:33.09 So what you're seeing in this cartoon
00:34:35.06 is our best guess, today,
00:34:37.12 of what the assembly line looks like.
00:34:40.07 There's a Module 1,
00:34:42.08 denoted as M1,
00:34:44.03 followed by a Module 2,
00:34:46.12 and you're seeing a zigzag type of a structure
00:34:51.05 that gives you a sense of what this 2 million Dalton
00:34:53.18 assembly line looks like.
00:34:56.00 So I will stop over here.
00:34:58.07 Hopefully this gives you a basic overview
00:35:00.09 of what these assembly line polyketide synthases are,
00:35:05.23 what chemistry they do,
00:35:07.16 and what they look like,
00:35:09.11 or at least our best guess, today,
00:35:11.04 of what they look like.
00:35:13.02 In subsequent modules,
00:35:14.13 we'll talk about other aspects of
00:35:17.10 these remarkable machines in nature.
00:35:19.16 Thank you.
- What is significant about the way polyketide antibiotics are synthesized? Why might such a system have evolved in nature?
- Why would it be useful to understand the individual components of polyketide biosynthesis assembly lines?
- What do all of the DEBS modules have in common? What implication does this have for the study of DEBS?
- What techniques have been used to analyze the structures of polyketide assembly lines like DEBS? What advantages/disadvantages do this different techniques have? How could structural models be useful?
- If you were to join the field of polyketide assembly lines, what would you want to study?
00:00:09.18 My name is Chaitan Khosla
00:00:11.09 and I'm a professor at Stanford University,
00:00:15.19 and this is part two
00:00:18.01 of the trilogy of my lectures
00:00:19.28 on assembly line polyketide biosynthesis.
00:00:24.11 In the previous lecture
00:00:25.27 I introduced you to the evolutionary biology
00:00:30.15 of these remarkable assembly lines,
00:00:33.00 the chemistry that happens
00:00:34.10 on these assembly lines,
00:00:36.07 and I gave you a general idea
00:00:38.05 of what these assembly lines look like.
00:00:42.02 So, we looked at the
00:00:44.12 6-Deoxyerythronolide B,
00:00:47.03 or DEBS,
00:00:50.11 that is responsible for making
00:00:52.23 this precursor of erythromycin.
00:00:57.21 I ended the previous lecture
00:01:00.15 by giving you a sense of what
00:01:02.18 we think this assembly line looks like
00:01:04.22 and how that insight was derived.
00:01:08.21 What I'm gonna start this module with
00:01:12.02 is an introduction to the kinds of tools
00:01:14.24 we use to interrogate
00:01:17.01 the biochemistry
00:01:19.06 of these remarkable assembly lines.
00:01:25.13 these assembly lines exist,
00:01:27.27 as we discussed in the first lecture,
00:01:30.22 in relatively esoteric sources.
00:01:33.17 They usually come from bacteria
00:01:35.22 whose names many of us have a hard time spelling,
00:01:39.11 or sometimes even worms,
00:01:41.25 or often times just sequence information
00:01:45.04 that was derived from DNA
00:01:47.04 that was collected from some place.
00:01:50.16 In order to study these systems,
00:01:53.07 one of the first sets of tools
00:01:55.14 that we developed
00:01:57.24 was to be able to take these
00:01:59.15 very complex metabolic pathways,
00:02:02.12 like the DEBS metabolic pathway,
00:02:04.26 and put them into
00:02:08.00 genetics-friendly microorganisms;
00:02:10.17 hosts like E. coli.
00:02:13.21 So, today you can make
00:02:16.20 6-Deoxyerythronolide B
00:02:19.15 in E. coli
00:02:21.07 by growing it in the presence of
00:02:24.12 glucose as a source of energy
00:02:26.11 and propionic acid
00:02:28.00 as a source of all the carbon
00:02:30.05 that's used to make the product,
00:02:32.04 so long as the recombinant E. coli
00:02:34.11 contains DNA
00:02:36.26 that instructs for the biosynthesis
00:02:39.10 of those three very large proteins
00:02:42.09 that comprise DEBS.
00:02:44.18 This is,
00:02:46.16 for obvious reasons,
00:02:48.18 a very powerful tool
00:02:50.15 to interrogate DEBS
00:02:52.06 because, now, if I have a bacterium
00:02:54.29 that makes my product for me,
00:02:57.09 I can go in there
00:02:59.15 for the price of a $200 kit,
00:03:02.12 manipulate the DNA
00:03:04.12 that encodes this assembly line
00:03:06.19 and ask,
00:03:08.06 what are the consequences of this assembly line?
00:03:11.03 And these tools have been
00:03:13.02 in our armamentarium
00:03:15.09 for the better part of the past two decades.
00:03:17.20 My previous generation of lectures
00:03:20.01 talked quite a bit about these tools,
00:03:22.11 so I won't spend a lot of time
00:03:24.01 doing so again,
00:03:26.05 but these tools
00:03:28.12 have played a critical role
00:03:30.03 in our understanding
00:03:31.21 of the biochemistry
00:03:33.01 of these assembly lines.
00:03:34.17 Now, what is more challenging,
00:03:37.12 but is perhaps arguably more important is,
00:03:41.28 if you want to study this remarkable enzymatic assembly line,
00:03:46.01 you'd like to be able to peel off the wrapper
00:03:49.28 that surrounds these remarkable proteins.
00:03:53.08 That is easier said than done,
00:03:55.20 but today, we can reconstitute
00:03:58.20 the entire 6-Deoxyerythronolide B synthase
00:04:02.15 from purified proteins.
00:04:05.06 What I show you on the lower-left corner
00:04:09.09 is a protein gel, an SDS-PAGE,
00:04:12.27 that shows five proteins.
00:04:17.20 The two proteins to the far right
00:04:21.01 are the second
00:04:23.23 and the third protein
00:04:26.15 of the erythromycin assembly line.
00:04:30.20 And each of them, as you can tell,
00:04:33.12 has a monomeric molecular mass
00:04:36.01 that exceeds 300 kilodaltons.
00:04:40.01 The third protein,
00:04:41.22 which is the first of these proteins,
00:04:45.03 could not be expressed
00:04:46.23 for love or money
00:04:48.14 in E. coli
00:04:50.13 in a form that gave adequate yields
00:04:53.07 of pure protein
00:04:54.28 to study biochemically,
00:04:56.28 and so we had to break it up
00:04:58.26 into three pieces,
00:05:01.04 which are shown in the
00:05:02.24 first three [lanes] of this gel,
00:05:05.14 and purify those pieces independently.
00:05:10.08 And now you can put those three pieces
00:05:13.14 together with the other two proteins
00:05:16.03 to make a cocktail of proteins
00:05:19.25 that, in the presence of appropriate substrates
00:05:23.06 -- propionyl coenzyme A,
00:05:27.04 and we don't use methylmalonyl coenzyme A itself,
00:05:31.01 instead we use an in situ enzymatic generation method
00:05:35.22 for methylmalonyl coenzyme A
00:05:38.00 where we use free methylmalonic acid,
00:05:41.01 coenzyme A,
00:05:42.24 and an enzyme called malonyl-CoA synthetase --
00:05:46.07 and so when you put these five proteins,
00:05:49.14 which have been purified,
00:05:51.13 together with all these precursors
00:05:53.26 in a test tube,
00:05:55.21 you see 6-Deoxyerythronolide B.
00:05:58.24 And what I show you on the lower right
00:06:01.07 is a mass spectrum of the product
00:06:04.00 that has been synthesized
00:06:05.29 in a biochemical equivalent
00:06:08.02 of an earth/air/water/fire type
00:06:10.19 of an experiment.
00:06:12.16 What this allows us now to do
00:06:14.24 is to probe this machine
00:06:17.05 with all the power
00:06:19.19 that you're used to using
00:06:21.24 to study your favorite enzyme
00:06:24.27 once you've purified it to homogeneity.
00:06:28.06 So what I show you in this
00:06:30.13 is a very simple graph
00:06:33.05 that gives you a sense
00:06:35.03 that we can turnover
00:06:36.26 this entire assembly line
00:06:38.27 in a test tube,
00:06:40.18 with a rate constant
00:06:42.15 that's approximately about 1/min.
00:06:47.10 So, approximately once every minute
00:06:49.17 this assembly line is releasing
00:06:51.21 6-Deoxyerythronolide B
00:06:53.22 in a test tube that is presented with the appropriate precursors,
00:06:57.27 and that is roughly the rate
00:06:59.24 we might expect this assembly line
00:07:01.18 to be working at
00:07:03.14 inside a cell.
00:07:05.06 I also wanna point out,
00:07:06.24 as the inset shows,
00:07:08.21 this assay is remarkably efficient.
00:07:12.29 Every equivalent of 6-Deoxyerythronolide B
00:07:16.28 has stoichiometric mapping
00:07:19.17 to an equivalent
00:07:21.26 of the propionyl-CoA primer
00:07:24.01 that is used,
00:07:25.28 and uses six equivalents of NADPH,
00:07:29.08 whose consumption is being measured
00:07:31.05 in this simple spectrophotometric assay.
00:07:35.07 Okay, so we have to tools to be able to study
00:07:39.04 the entire assembly line
00:07:40.25 inside a recombinant E. coli-like cell.
00:07:44.06 We have the ability
00:07:46.00 to study the entire assembly line
00:07:48.01 in a purified, reconstituted form.
00:07:51.03 We also have the ability, today,
00:07:53.21 to study the individual steps
00:07:56.12 in the catalytic cycle of these modules
00:08:01.09 in isolation.
00:08:03.10 So, recall in the previous module,
00:08:05.29 I introduced you to some of the
00:08:08.12 core reactions
00:08:10.18 that occur at every module.
00:08:13.00 There's a reaction
00:08:14.19 that we call chain translocation,
00:08:16.24 where the chain moves
00:08:18.08 from the acyl carrier protein
00:08:20.04 in the upstream module
00:08:21.26 to the module that's receiving the chain,
00:08:25.09 and if we want to interrogate
00:08:27.07 just that reaction
00:08:29.15 for one module,
00:08:31.09 what we do is pull out that module
00:08:34.07 from the rest of the assembly line,
00:08:36.17 purify that to homogeneity,
00:08:39.23 present it with
00:08:43.01 a chemoenzymatically-derived acyl carrier protein
00:08:47.00 that has the growing polyketide chain substrate
00:08:51.01 bound to it,
00:08:53.03 and we put it into a test tube
00:08:55.19 so that the chain translocation event
00:08:58.13 -- the movement of that growing polyketide chain
00:09:01.23 into the module --
00:09:03.26 is the slow kinetic step,
00:09:06.00 and everything after that
00:09:08.03 that leads to the turnover of this module
00:09:10.21 is fast.
00:09:12.08 And so you can use
00:09:14.07 this kind of an assay
00:09:15.28 to interrogate,
00:09:18.01 using established kinetic paradigms,
00:09:20.23 that chain translocation step
00:09:23.10 of your favorite module
00:09:25.12 that you're interested in.
00:09:27.22 The same approach can also be used
00:09:31.03 to kinetically isolate the chain elongation event
00:09:34.11 that I introduced you to
00:09:36.03 in the earlier lecture.
00:09:38.18 So when we want to study chain elongation,
00:09:42.03 what we do is we take the module
00:09:44.22 whose elongation biochemistry
00:09:46.19 we want to study,
00:09:48.19 and we prepare just the ketosynthase
00:09:51.03 together with the acyltransferase
00:09:54.08 from that module
00:09:56.02 as one protein.
00:09:57.21 We produce its carrier protein,
00:10:00.04 its acyl carrier protein,
00:10:01.29 as another protein.
00:10:03.27 We put these two proteins together,
00:10:06.21 we present the two substrates
00:10:09.24 into this assay,
00:10:12.05 and we look for chain elongation,
00:10:15.00 which gives rise to the product.
00:10:17.19 And we do this under conditions
00:10:20.06 where the step we wanna probe,
00:10:22.07 the elongation step,
00:10:23.27 is the slow step, and everything else is fast.
00:10:27.20 You can do exactly the same thing
00:10:29.21 to probe the acyl transfer,
00:10:31.29 the selection of that building block
00:10:34.16 -- methylmalonyl coenzyme A-derived building block
00:10:38.12 from metabolism --
00:10:40.08 the same approach can also work over there.
00:10:42.20 And all of these assays are well-developed,
00:10:44.29 they're in the literature,
00:10:46.19 and you can use them to study
00:10:48.08 your favorite assembly line.
00:10:51.07 In addition to those core reactions
00:10:53.28 -- chain translocation,
00:10:55.21 chain elongation,
00:10:57.08 and acyl transfer --
00:10:58.23 I mentioned there are auxiliary reactions,
00:11:01.13 which I lumped under chain modification.
00:11:04.27 Those reactions include
00:11:06.22 ketoreductase-types of chemistries.
00:11:10.11 In this particular assay,
00:11:12.11 I'm adding the ketoreductase,
00:11:15.03 or KR,
00:11:16.26 as a stand-alone protein,
00:11:18.16 to the rest of my system
00:11:21.04 so that I can control the rate
00:11:22.27 at which that step occurs,
00:11:24.27 and I can look at the consequences
00:11:27.13 of putting one ketoreductase in my assay
00:11:30.04 as opposed to some other ketoreductase.
00:11:33.04 And that allows me to interrogate
00:11:35.28 the ketoreductase reaction.
00:11:38.00 You can do the same thing
00:11:40.04 at the level of the dehydratase reaction,
00:11:43.15 which follows after the ketoreductase reaction
00:11:47.29 in certain chain modification sequences.
00:11:52.00 So, all of these assays are also set up.
00:11:54.22 The point you need to recognize is that
00:11:57.13 you can probe through, again,
00:11:59.04 a divide-and-conquer approach,
00:12:01.00 the chemistry happening at any one of these steps
00:12:05.05 in the overall assembly line.
00:12:08.22 Using this combination of in vivo and in vitro tools,
00:12:14.02 there are a number of important problems
00:12:16.15 you can study.
00:12:17.28 In the remainder of this second module lecture,
00:12:21.16 I will talk to you about some examples
00:12:24.13 of questions, long-standing questions
00:12:26.26 in the field,
00:12:28.04 having to do with the specificity
00:12:29.28 of these assembly lines.
00:12:31.20 I'll give you two examples of those problems
00:12:33.22 because they have engineering implications.
00:12:36.21 And then in the next lecture we'll talk about
00:12:40.08 the assembly line mechanisms.
00:12:42.27 So, stereospecificity
00:12:46.01 is probably one of the most fascinating features
00:12:50.15 of these complex polyketide antibiotics
00:12:53.11 that these assembly lines make.
00:12:55.24 So, to the right,
00:12:57.14 you're seeing the 6-Deoxyerythronolide B product
00:13:01.16 of DEBS,
00:13:03.07 and for those of you who are looking at that now,
00:13:05.22 you're noticing that it has 10 stereocenters.
00:13:12.02 That is 2^10 possible chiral forms
00:13:17.08 of the same chemical formula,
00:13:20.01 or slightly more than 1000
00:13:22.12 of these chiral forms.
00:13:24.20 If you go to a fermentation plant
00:13:27.10 that makes erythromycin,
00:13:30.08 the large vat that produces erythromycin
00:13:33.23 has one out of those 1000+
00:13:38.04 stereochemical forms in it;
00:13:40.17 the one that I'm showing you.
00:13:43.00 I think most of you would recognize
00:13:45.01 that that is a really impressive feat
00:13:48.04 on the part of nature...
00:13:49.27 how it can program this assembly line
00:13:52.06 to give one, and only one
00:13:54.13 stereochemical outcome.
00:13:56.22 That is a problem
00:13:58.29 that we have quite a good understanding of
00:14:02.16 how that happens today.
00:14:05.08 I've cited some references on this slide,
00:14:09.04 and so I will summarize for you
00:14:11.07 what these references teach us
00:14:13.18 about how stereochemistry is controlled
00:14:17.07 by the DEBS assembly line.
00:14:20.00 So, of those 10 stereocenters,
00:14:25.00 one of them,
00:14:27.12 which is this stereocenter,
00:14:31.06 is generated by
00:14:34.28 this ketoreductase
00:14:37.28 in Module 3 of the assembly line,
00:14:40.19 that I introduced to you as that epimerase,
00:14:44.29 that looks like a ketoreductase
00:14:46.29 in my previous lecture.
00:14:49.06 This is the enzyme that is a homologue
00:14:52.02 of other ketoreductases,
00:14:53.18 but does no NADPH-dependent chemistry.
00:14:57.27 Instead, it epimerizes the C2 carbon atom
00:15:01.20 of the growing polyketide chain
00:15:04.28 that is lodged in Module 3
00:15:06.25 of the assembly line.
00:15:09.05 Of the remaining 9 stereocenters,
00:15:12.07 8 of those stereocenters
00:15:14.22 are shown in red,
00:15:17.09 and they are controlled
00:15:19.21 by the 3 red ketoreductases
00:15:23.02 and 1 blue ketoreductase
00:15:25.17 in Modules 1, 2, 5, and 6, respectively.
00:15:32.23 So, each of these 4 ketoreductases
00:15:37.25 controls 2 stereocenters apiece.
00:15:42.20 For the chemically initiated,
00:15:44.29 these enzymes are not just stereoselective,
00:15:48.22 they're also diastereoselective;
00:15:51.17 so they're setting 2 stereocenters at a time.
00:15:56.01 And these enzymes, we know...
00:15:58.23 these 4 enzymes we know, today,
00:16:01.13 are both necessary and sufficient
00:16:04.10 for the unique labeling...
00:16:08.05 for the unique identification
00:16:11.01 of those stereocenters.
00:16:13.21 The last stereocenter,
00:16:16.01 which is this stereocenter,
00:16:19.13 is at the 6 position,
00:16:22.01 is a more complex output,
00:16:25.08 and it is generated by 3 enzymes
00:16:27.28 in Module 4 of DEBS.
00:16:30.28 There is a ketoreductase,
00:16:34.03 a dehydratase,
00:16:36.04 and an enoylreductase,
00:16:38.08 that all collaborate with each other
00:16:41.06 to set this one stereocenter.
00:16:47.06 In addition to stereochemistry,
00:16:49.20 there is another very important
00:16:53.15 specificity that is encoded
00:16:56.11 in this assembly line
00:16:58.22 at each module,
00:17:00.24 and that is the specificity
00:17:05.17 that corresponds to the choice
00:17:08.19 of the extender unit.
00:17:10.19 In my introduction,
00:17:12.07 I pointed out that all of the modules
00:17:14.26 of 6-Deoxyerythronolide B synthase
00:17:18.04 use a methylmalonyl coenzyme A
00:17:21.14 extender unit.
00:17:23.22 In the case of DEBS,
00:17:27.06 the R group that is shown
00:17:29.25 in this enzymatic scheme
00:17:32.07 would be a methyl group.
00:17:34.17 Other polyketide synthases
00:17:37.06 can use coenzyme A thioesters
00:17:39.29 that contain other functional groups
00:17:42.16 in place of a methyl group, over here.
00:17:46.11 And all of these choices
00:17:48.18 are made by the acyltransferase.
00:17:53.00 These acyltransferases
00:17:55.10 are relatively specific.
00:17:58.01 Not only do they have high specificity,
00:18:01.04 as shown in this graph
00:18:03.15 for the coenzyme A precursor
00:18:05.28 they're picking from the metabolic soup...
00:18:09.09 so what you see in this graph over here
00:18:13.06 is the rate...
00:18:15.24 the velocity versus substrate concentration
00:18:18.20 of the preferred substrate,
00:18:20.20 which is methylmalonyl coenzyme A,
00:18:23.22 and down here are the rates
00:18:25.25 if R is one methyl short,
00:18:28.13 so in other words it's a hydrogen instead of a methyl,
00:18:31.14 or one methyl longer,
00:18:33.23 which is an ethyl group.
00:18:35.29 And as you can tell,
00:18:37.25 this acyltransferase
00:18:39.18 that we're showing you data for in this slide
00:18:42.08 is highly selective
00:18:44.24 for a methyl group
00:18:46.25 instead of one smaller or one larger.
00:18:50.16 Now, in addition to being specific
00:18:52.25 for its cognate substrate,
00:18:56.09 this enzyme is also specific
00:18:59.11 for its protein partner,
00:19:01.09 which is the acyl carrier protein,
00:19:03.22 that is being used.
00:19:05.25 And here I'm introducing you
00:19:07.25 to a concept that is gonna come back
00:19:10.11 in a more significant way
00:19:12.13 in the last of my three lectures,
00:19:14.16 which is the importance
00:19:16.10 of protein-protein interactions
00:19:19.04 in the assembly line biochemistry
00:19:22.00 of these systems.
00:19:23.22 In this case, what you're seeing is
00:19:27.03 that the acyl carrier protein
00:19:29.21 is being strongly recognized
00:19:32.00 by the acyltransferase,
00:19:34.05 because if you give this same acyltransferase
00:19:37.18 other acyl carrier proteins
00:19:39.25 from other modules of DEBS
00:19:41.23 or elsewhere,
00:19:43.19 they work much more poorly
00:19:46.09 than the natural acyl carrier protein.
00:19:49.19 So in addition to recognizing
00:19:51.15 the coenzyme A precursor,
00:19:53.27 you also have recognition
00:19:56.00 of the acyl carrier protein.
00:19:58.18 Now, for those of you who are familiar
00:20:00.16 with enzymes kinetics
00:20:02.16 know that from data like this,
00:20:04.18 you can derive mechanisms
00:20:06.10 of how these enzymes work.
00:20:08.15 So, in this case,
00:20:10.19 this acyltransferase
00:20:13.02 has a ping-pong
00:20:15.03 bi-bi-type of a mechanism.
00:20:18.11 The coenzyme A precursor first comes in,
00:20:21.21 it is bound by the acyltransferase,
00:20:25.05 the acyltransferase picks the methylmalonyl extender unit,
00:20:29.02 coenzyme A leaves,
00:20:30.23 the carrier protein comes in,
00:20:32.29 is recognized by the acyltransferase,
00:20:35.25 and takes away the product
00:20:38.06 - the methylmalonyl extender unit.
00:20:40.23 So that ping-pong element comes into
00:20:43.12 this kind of a mechanism.
00:20:45.12 Now, you can also ask,
00:20:46.29 in addition to these gatekeeper acyltransferases
00:20:50.25 that control the choice of the building block,
00:20:55.18 there are many other enzymes
00:20:57.18 in this assembly line
00:20:59.17 that lie downstream of each choice
00:21:02.15 that a module makes
00:21:04.16 of its building block.
00:21:06.09 To what extent do they influence
00:21:09.05 the overall substrate specificity?
00:21:11.29 Do they care about what the upstream module chose
00:21:16.17 as its precursor for elongating
00:21:20.01 the growing polyketide chain?
00:21:22.13 We can ask questions like that
00:21:24.16 using the assays... biochemical assays
00:21:27.11 I showed you earlier on,
00:21:29.16 and from those experiments you learn something
00:21:31.22 quite interesting.
00:21:33.16 So, you learn that the downstream steps,
00:21:36.14 beyond the acyltransfer step,
00:21:38.28 in many modules
00:21:41.12 are not that discriminatory,
00:21:43.09 analogous to what Henry Ford
00:21:45.11 had contemplated for his assembly line.
00:21:48.26 The downstream steps
00:21:51.01 have low, but not a significant amount,
00:21:54.16 of specificity.
00:21:56.21 So if, by whatever mechanism,
00:21:59.14 you can fool this acyltransferase
00:22:02.25 to put a hydrogen instead of a methyl
00:22:06.16 at this position on the carrier protein,
00:22:09.26 the elongation enzyme
00:22:12.06 that elongates the chain
00:22:14.10 and puts this R group in the growing polyketide chain,
00:22:19.14 primarily loses
00:22:22.12 about 2- to 4-fold specificity
00:22:25.10 as a result of this mistake
00:22:28.13 that the upstream step made.
00:22:31.00 That's not much in the grand scheme of things,
00:22:33.25 but what you have to remember is
00:22:37.02 these assembly lines have
00:22:39.18 many, many downstream steps.
00:22:41.27 So, one of these assembly lines,
00:22:43.23 the first module over here, or the second module,
00:22:46.28 has four or five modules downstream
00:22:50.17 that are looking at the consequences
00:22:53.01 of what that module did.
00:22:55.00 And so these small effects
00:22:57.09 at the level of substrate specificity
00:22:59.23 then have quite significant impact
00:23:02.21 on the final product,
00:23:04.18 and you can see this
00:23:06.18 in the context of assays like this.
00:23:09.06 So here,
00:23:10.24 in the spirit of engineering,
00:23:12.17 what we're doing is we're taking that natural...
00:23:15.17 the natural assembly line that nature uses
00:23:18.04 to make 6-Deoxyerythronolide B,
00:23:20.27 we're taking that full assembly line,
00:23:23.00 and instead of just presenting it
00:23:25.27 methylmalonyl coenzyme A,
00:23:28.14 we're now presenting it a 1:1 mixture
00:23:32.06 of methylmalonyl coenzyme A
00:23:34.11 and ethylmalonyl coenzyme A,
00:23:36.28 and we're asking what's gonna happen.
00:23:40.11 Are you gonna get just 6-Deoxyerythronolide B?
00:23:44.19 Are you gonna get something else,
00:23:47.10 one or two other products?
00:23:49.06 Or are you gonna get a zoo of products?
00:23:51.26 And the answer to that is,
00:23:53.28 you get some analogues
00:23:56.23 that are produced competitively
00:23:58.25 with the natural product 6-DEB,
00:24:02.23 but these compounds
00:24:05.24 aren't immediately obvious
00:24:08.00 why these should be formed
00:24:10.02 and other ones shouldn't be formed.
00:24:11.29 So at least one of these, for example,
00:24:13.27 this peak that I show you out here,
00:24:16.04 whose mass spectrum is shown over here,
00:24:18.22 we know with reasonable confidence,
00:24:21.06 has an ethyl group
00:24:23.07 that is incorporated at the C8 position
00:24:26.12 instead of a methyl group.
00:24:28.06 So, somehow,
00:24:30.10 that gatekeeper transferase
00:24:32.29 has enough tolerance
00:24:35.04 for an ethylmalonyl extender unit,
00:24:37.24 and all of the downstream enzymes
00:24:40.13 on the assembly line
00:24:42.11 are sufficiently tolerant
00:24:44.13 that they will let that mistake slide by,
00:24:46.28 so you get this desired product.
00:24:50.05 And if we could predict
00:24:52.10 what's gonna be made and what's not gonna be made
00:24:55.01 through an experiment like this,
00:24:57.05 why, then, we would have a way to precisely engineer
00:24:59.21 an antibiotic like erythromycin
00:25:01.21 to make a molecule like this.
00:25:03.27 But right now, we're just beginning to scratch
00:25:06.10 the tip of the iceberg
00:25:07.22 in terms of what's possible,
00:25:09.13 and what's not,
00:25:11.04 in these kinds of systems,
00:25:12.21 and an experiment like this illustrates
00:25:14.22 what's possible.
00:25:16.28 These kinds of insights can also be used
00:25:19.12 for most sophisticated engineering experiments,
00:25:22.07 analogous to the kinds of experiments
00:25:24.17 you may be familiar with
00:25:26.25 when you think about incorporating unnatural amino acids
00:25:31.18 in proteins that are derived
00:25:33.24 by ribosomal mechanisms.
00:25:36.04 And so, in this,
00:25:38.08 there are some examples of acyltransferases
00:25:42.10 that are what we call stand-alone acyltransferases.
00:25:46.12 They operate outside the assembly line,
00:25:49.21 and because they work
00:25:51.29 very, very fast compared to typical assembly line acyltransferases
00:25:58.03 that exist in assembly lines,
00:26:00.16 you can do some interesting experiments.
00:26:02.25 So in this case,
00:26:04.24 we have knocked out
00:26:07.10 one acyltransferase
00:26:09.17 out of all the six acyltransferases
00:26:12.09 on the 6-Deoxyerythronolide B synthase
00:26:16.23 -- so this is like a site-directed mutant
00:26:19.14 that has inactivated that acyltransferase --
00:26:23.02 and we complement it,
00:26:25.15 in trans,
00:26:27.26 with that very ultra-fast acyltransferase
00:26:30.27 that we get from a different assembly line
00:26:34.02 in nature.
00:26:36.07 And what happens is,
00:26:38.00 because this acyltransferase
00:26:40.09 will pick a malonyl unit instead of a methylmalonyl unit,
00:26:44.13 it can transfer that malonyl unit
00:26:47.07 onto this module,
00:26:49.02 because this module is simply waiting
00:26:50.26 for something to come its way,
00:26:52.29 and this enzyme can do it pretty quickly,
00:26:56.18 and now that resulting intermediate
00:26:58.27 moves all the way down in the assembly line
00:27:01.23 to give you a product
00:27:04.09 that has one and only one change
00:27:07.11 in the entire macrocycle
00:27:10.09 that is made.
00:27:12.08 And what you see over here is,
00:27:13.29 if I have my assembly line that is present
00:27:18.15 at say micromolar concentrations in my assay,
00:27:22.00 at nanomolar concentrations
00:27:24.26 I can get quite respectable incorporation
00:27:29.22 of malonyl coenzyme A
00:27:31.18 at that single position,
00:27:33.16 to give me 12-desmethyl-6-Deoxyerythronolide B.
00:27:39.25 So this is a way you can cheat the system,
00:27:43.26 so long as you have
00:27:46.19 a robust enzyme
00:27:49.00 that can trans-complement the module
00:27:51.15 that you wanna cheat.
00:27:53.22 So hopefully that gives you
00:27:55.24 a flavor of the kinds of tools we have
00:27:58.28 and the way we use these tools
00:28:01.01 to study specificity.
00:28:03.12 Thank you.
- Why might a molecule like 6-deoxyerthronolide B need this type of assembly line instead of a standard enzymatic pathway?
- Why do you think adding other R groups (other than hydrogen and ethyl) to the growing polyketide chain was not discussed?
- What do you think is an interesting open question presented in this lecture and why?
00:00:08.03 Hello again.
00:00:09.20 I'm Chaitan Khosla
00:00:11.11 from Stanford University,
00:00:13.10 and welcome to the third part
00:00:16.24 of this trilogy of lectures
00:00:18.29 on polyketide antibiotic biosynthesis
00:00:22.05 and assembly lines.
00:00:25.29 So, in the first lecture
00:00:27.21 I introduced you to the basics
00:00:30.02 of what an assembly line is,
00:00:33.13 what's the chemistry that happens on an assembly line,
00:00:37.03 and what an assembly line looks like.
00:00:41.15 We looked at the basic architecture
00:00:45.06 of the 6-Deoxyerythronolide B synthase,
00:00:48.19 or DEBS,
00:00:50.18 which I have used subsequently
00:00:52.27 to make most of the points
00:00:55.08 that I have made.
00:00:57.12 I introduced to you
00:00:59.16 the architecture of this assembly line,
00:01:01.25 or at least as best as we can gauge today
00:01:05.12 based on data we have.
00:01:07.24 In the second lecture
00:01:10.14 I introduced you to the tools,
00:01:13.09 and how we've used those tools
00:01:15.24 to study different kinds of questions.
00:01:18.23 So, we talked about refactoring
00:01:21.18 a metabolic pathway
00:01:23.24 like this assembly line of DEBS
00:01:26.03 in E. coli.
00:01:27.09 We talked about how to reconstitute that pathway
00:01:30.07 in a test tube.
00:01:32.02 I pointed out that you can
00:01:33.25 isolate individual reactions
00:01:36.12 on the assembly line
00:01:38.05 and look at the kinetics,
00:01:39.22 whether you're looking at extender unit selection,
00:01:44.27 modification events.
00:01:46.24 We also talked about how these tools
00:01:48.23 can be used to study specificity
00:01:51.03 and, in particular,
00:01:52.29 we talked about two types of specificities:
00:01:58.24 I mentioned that stereochemistry
00:02:01.06 is controlled by reductive domains,
00:02:03.23 as well as extender unit specificity,
00:02:07.17 and I mentioned, over there,
00:02:09.25 you not only have enzyme-substrate recognition
00:02:13.15 but also protein-protein recognition.
00:02:18.04 This final lecture focuses on what, in my mind,
00:02:22.10 is the grandest challenge of all
00:02:25.03 in this field:
00:02:27.05 understanding how an assembly line
00:02:30.17 actually comes about.
00:02:33.20 What do I mean by that?
00:02:37.05 I've been showing you,
00:02:39.06 quite freely I might add,
00:02:41.00 schemes like this
00:02:43.08 throughout my earlier two lectures.
00:02:47.20 Every time I show you one of these schemes
00:02:50.08 or, for that matter,
00:02:52.04 you could open your favorite journal of biochemistry today,
00:02:55.26 and chances are in any given issue,
00:02:58.07 you'll find somebody having published
00:03:00.14 yet another one of these
00:03:02.13 kinds of assembly lines
00:03:04.04 that they have cloned, sequenced,
00:03:05.21 and started to characterize.
00:03:07.07 And in all cases you'll find
00:03:09.01 cartoons like the kind you see in this scheme.
00:03:15.26 The question that lies ahead of us is,
00:03:18.11 how do we know that
00:03:21.02 this is how the assembly line works?
00:03:26.27 And one way to recognize
00:03:29.12 why that question
00:03:31.25 is not an obvious question
00:03:34.15 is by thinking back
00:03:36.08 to all those orphan polyketide assembly lines
00:03:39.23 that I talked about to you
00:03:41.27 at the beginning of my first lecture.
00:03:43.29 So, I mentioned to you,
00:03:45.29 there's these large numbers of orphan assembly lines
00:03:49.03 that make polyketides
00:03:51.10 whose structure we don't know.
00:03:54.14 If I gave you the sequence
00:03:57.02 of one of those assembly lines
00:03:59.17 and asked you
00:04:01.17 to draw me an equivalent scheme
00:04:03.06 like the kind you see in front of you
00:04:05.02 in this slide,
00:04:07.10 chances are you won't be able to do it
00:04:09.15 with any confidence.
00:04:11.26 And the reason why I can show
00:04:15.07 this scheme in this slide,
00:04:16.24 and hold my head up high,
00:04:19.03 is because I know the answer
00:04:23.07 for this assembly line.
00:04:25.26 I know the structure
00:04:27.20 of 6-Deoxyerythronolide B,
00:04:30.13 and using that knowledge I am essentially
00:04:34.14 back-programming on this assembly line
00:04:37.08 what must happen at each stage
00:04:40.07 in order to get that final product.
00:04:43.20 But that begs the question,
00:04:45.27 how does nature know
00:04:48.01 that this is how the assembly line must work?
00:04:50.27 Why does nature start here,
00:04:54.01 and move here?
00:04:55.29 Or putting it differently,
00:04:57.22 why can't nature take a product
00:05:00.09 from this module
00:05:02.09 and instead of passing it on to this module,
00:05:05.12 why can't it just skip these modules altogether
00:05:08.21 and pass it on to this?
00:05:10.29 Or, why can't it take this precursor
00:05:15.13 and pass it to some other polyketide synthase
00:05:18.05 that's floating around in the same cell?
00:05:21.20 Where are the rules
00:05:24.17 that dictate how this vectorial translocation
00:05:28.17 of polyketides
00:05:30.15 must occur in the manner
00:05:32.11 that I tell you it's occurring
00:05:34.20 in this scheme?
00:05:36.20 That is the fundamental challenge
00:05:39.21 associated with this field.
00:05:43.27 The way we think about this challenge
00:05:46.23 goes back to the work
00:05:50.08 of an inspirational enzymologist
00:05:54.26 from a previous generation,
00:05:56.16 whose name you may have heard:
00:05:58.25 Bill Jencks, or William P. Jencks.
00:06:02.29 Jencks made huge contributions
00:06:06.07 to our understanding
00:06:08.08 of how enzymes work.
00:06:10.15 One of the problems he talked about
00:06:12.29 and gave a lot of thought to,
00:06:15.09 as is discussed in this article
00:06:17.07 that I would encourage any student
00:06:19.08 who's interested in machines in biology
00:06:24.03 to read,
00:06:25.21 was the question of how do you get
00:06:28.24 vectorial processes occurring in nature,
00:06:32.19 off the back of proteins like enzymes?
00:06:37.13 This article is chock-full
00:06:40.05 of many really interesting insights
00:06:42.21 that were way ahead of their time.
00:06:46.01 I certainly don't have the time
00:06:47.27 to go into all of them.
00:06:50.03 But there's one insight
00:06:51.27 that I'm going to focus on
00:06:53.21 because I think it does a very nice job
00:06:55.27 of framing the understanding
00:06:58.11 of the vectorial chemistry
00:07:00.05 that I'm here to tell you about.
00:07:02.26 What Jencks says in this article
00:07:07.03 is that proteins that do coupled vectorial processes,
00:07:10.27 that do vectorial processes,
00:07:13.06 differ from your garden variety enzyme
00:07:16.13 in the sense that they undergo large changes
00:07:21.08 in substrate specificity
00:07:23.14 at different points in their overall catalytic cycle.
00:07:29.26 And a really good example
00:07:32.14 that he uses in his quintessential
00:07:35.01 chicken-scratch cartoon manner
00:07:37.27 that's shown over here
00:07:40.08 is the example of how
00:07:43.03 myosin moves on actin fibers.
00:07:46.15 That you probably know about
00:07:49.00 because it's what textbooks are made up...
00:07:51.09 biochemistry textbooks are made of.
00:07:53.18 What you know is that myosin
00:07:56.07 moves along an actin highway
00:07:59.16 that is made up of actin monomers
00:08:02.15 by essentially going from one monomer of actin
00:08:07.15 to the next and so on.
00:08:11.07 And that movement
00:08:13.18 is powered by the hydrolysis of ATP,
00:08:17.19 by myosin.
00:08:19.28 And what Jencks postulated back then,
00:08:22.14 or what he articulated in this paper,
00:08:25.28 was that the reason you get this vectorial movement
00:08:32.07 is because there are different states of myosin
00:08:35.15 at different points in the overall cycle,
00:08:39.15 and some of these states
00:08:42.07 bind tightly to myosin,
00:08:44.22 the unbound states,
00:08:47.03 whereas ATP- or ADP-bound states,
00:08:50.19 the myosin does not bind very tightly
00:08:54.00 to actin.
00:08:56.21 The key point that Jencks made
00:08:59.14 is the reason this is a machine,
00:09:01.22 and it's not like myosin
00:09:03.23 dances back and forth on this actin highway,
00:09:07.09 the reason it motors along this actin highway
00:09:10.25 is because there is a distinct change
00:09:14.27 in specificity of myosin for actin
00:09:18.14 at different points in the catalytic cycle.
00:09:21.21 When myosin binds to ATP,
00:09:26.01 it exchanges with an unbound state
00:09:32.03 that binds to one monomer of actin,
00:09:36.02 whereas when that ATP is hydrolyzed,
00:09:39.23 that myosin sets up the binding
00:09:44.09 of a distinct conformational state
00:09:46.29 to the next.
00:09:49.03 It can't...
00:09:51.11 this exchange between the bound and the unbound state
00:09:54.29 is prohibited, because if this happened
00:09:57.03 you would be wasting this ATP hydrolysis,
00:10:00.21 and you'd be back to where you started from.
00:10:04.01 But the reason this moves forward
00:10:06.20 is because this state
00:10:09.00 has a distinct specificity
00:10:12.23 than this state of the enzyme.
00:10:15.27 So the big changes
00:10:19.02 in the specificity of myosin
00:10:21.13 are what Jencks proposed
00:10:23.12 is the basis for this vectorial process.
00:10:28.02 We think about our challenge
00:10:32.06 very much analogous
00:10:34.17 to how Jencks proposed
00:10:37.13 how myosin works on actin.
00:10:39.29 And more specifically,
00:10:42.10 there are two postulates
00:10:44.20 that we draw upon
00:10:46.28 from Jencks' analogy
00:10:49.05 to actin and myosin
00:10:51.07 to explain how this vectorial process happens.
00:10:55.08 The first one
00:10:57.19 is that the specificity change
00:11:00.14 that Jencks talked about,
00:11:03.05 in our context,
00:11:05.23 happens at the level of protein-protein interactions.
00:11:09.26 I gave you an example
00:11:12.04 in my previous lecture
00:11:14.04 of an acyltransferase
00:11:16.21 that has protein-protein specificity
00:11:19.14 in addition to its specificity
00:11:21.24 for its normal substrate.
00:11:23.18 I'll be elaborating on this theme
00:11:25.19 much more in this lecture.
00:11:28.07 And the second hypothesis
00:11:31.09 that helps us think about assembly line
00:11:34.20 vectorial biochemistry
00:11:37.03 is that this core of the protein
00:11:39.22 that exists in each module,
00:11:41.20 this KS and AT,
00:11:44.26 has specificity for the acyl carrier protein,
00:11:50.15 in an interesting way,
00:11:52.20 that leads to establishing directionality.
00:11:55.26 Bear with me if that's not clear.
00:11:58.10 Let me first start by giving you
00:12:00.14 some information, some background
00:12:02.18 on the relevance of protein-protein interactions.
00:12:05.17 So, about 15 years ago,
00:12:09.07 when Rajesh Gokhale was in my lab,
00:12:12.08 he came up with an interesting postulate
00:12:15.27 about how assembly line directionality happens.
00:12:21.02 He hypothesized
00:12:22.27 that specificity that leads to this chain
00:12:26.07 moving from this module to this module
00:12:29.08 is not in the active sites
00:12:32.12 for different substrates,
00:12:35.04 but lies at the interface
00:12:37.18 between these modules.
00:12:39.22 So you have these docking sites
00:12:43.08 that exist between modules,
00:12:46.02 that are orthogonal
00:12:48.13 to the core modules
00:12:50.22 that are doing the actual enzymology,
00:12:53.19 that allow two modules
00:12:56.05 to come together
00:12:58.04 so that you have this directionality
00:13:01.05 that is established.
00:13:03.03 How do we know that?
00:13:04.28 We know that through experiments like this.
00:13:07.19 This is one example of an experiment
00:13:09.14 that Stuart Tsuji did in my lab
00:13:11.26 some years ago
00:13:14.08 where he kinetically isolated,
00:13:16.25 from the entire DEBS assembly line
00:13:19.28 you see over here,
00:13:22.03 these two modules,
00:13:24.00 Module 2 and Module 3.
00:13:25.21 He pulled them out of the overall system,
00:13:28.09 put them into a test tube,
00:13:30.09 and reconstituted their full enzymology
00:13:33.16 that's shown in this scheme,
00:13:36.06 measuring the appropriate parameters
00:13:38.21 that one gets from these kinds of experiments.
00:13:43.05 Now, what he does is
00:13:45.19 he switches these short peptide linkers
00:13:48.11 that flank these modules
00:13:50.25 for their counterparts from this module.
00:13:54.29 And if he switches just one of the two linkers,
00:13:58.25 what he gets are two modules
00:14:01.18 that by themselves
00:14:03.25 are just as good of catalysts as they were here,
00:14:08.02 but when you put them together,
00:14:10.04 they are simply unable to talk to each other.
00:14:15.07 If, however,
00:14:16.26 you take this module that has this linker
00:14:19.08 instead of this linker,
00:14:21.25 and couple it to this module
00:14:26.23 that has this linker
00:14:28.29 instead of this linker,
00:14:31.12 this pair over here
00:14:33.18 is indistinguishable from this pair.
00:14:36.23 And so now you see
00:14:39.06 how these matching docking sites
00:14:43.22 provide for the ability for Module 2
00:14:47.02 to talk to Module 3,
00:14:49.20 as opposed to Module 5
00:14:51.23 or any of the other modules.
00:14:53.25 So you're seeing the establishing of directionality
00:14:57.14 off the back of protein-protein interactions.
00:15:02.27 Now, what the Gokhale/Tsuji experiments do
00:15:05.18 is they tell us why Module talks to Module 3.
00:15:09.21 What they don't tell us is,
00:15:12.01 why don't chains go back and forth?
00:15:15.00 It's analogous to the question
00:15:17.09 that Jencks was asking:
00:15:18.28 why is it that myosin moves
00:15:20.21 in only one direction on actin,
00:15:23.12 as opposed to just going back and forth,
00:15:25.19 back and forth?
00:15:27.20 That question has an exact analogy over here,
00:15:30.27 because as you'll remember from the first lecture
00:15:33.11 of my three lectures,
00:15:35.06 I introduced to you that chain translocation
00:15:38.09 -- when a chain moves from one module
00:15:40.02 to the next --
00:15:41.24 it does so by a thiol
00:15:43.27 to a thioester exchange reaction.
00:15:47.05 And that reaction
00:15:48.26 can just as easily run in the backward direction
00:15:51.03 as it can run in the forward direction.
00:15:53.23 And so what precludes chains
00:15:55.22 from just going back and forth
00:15:57.15 and messing up the chemistry
00:15:59.06 so you'll never get an antibiotic
00:16:00.24 out of these assembly lines?
00:16:02.22 What's written in this code
00:16:04.17 that allows that to happen?
00:16:09.05 So, the question is,
00:16:12.06 how does this event
00:16:14.25 lead to a movement here,
00:16:17.01 but when it comes time to move the chain forward,
00:16:20.14 it doesn't just go back
00:16:22.15 into the same module,
00:16:24.09 but rather goes to the next module.
00:16:29.12 Again, over here,
00:16:31.17 protein-protein interactions
00:16:33.19 play a very important role.
00:16:35.13 And in particular,
00:16:37.24 the interactions,
00:16:40.04 not just in these docking sites
00:16:42.00 that Gokhale and Tsuji identified,
00:16:43.29 but also between this protein
00:16:46.22 and this protein,
00:16:48.21 play a very critical role.
00:16:50.23 How do we know that?
00:16:52.17 Through experiments like this
00:16:54.11 that Nick Wu did,
00:16:56.06 where he isolated, kinetically,
00:16:58.02 this chain translocation event,
00:17:01.26 exposed this module over here
00:17:06.08 to the same substrate,
00:17:08.07 same docking partners,
00:17:10.15 same receiving module,
00:17:12.22 but now he changes
00:17:14.19 the identity of the donor carrier protein.
00:17:17.20 So all that's being changed over here
00:17:19.19 is the identity of this protein.
00:17:22.05 And what you see is
00:17:24.11 you have a big difference
00:17:26.19 in specificity constants
00:17:28.24 just based upon who this player is
00:17:32.19 in the overall chain translocation event,
00:17:35.11 suggesting that these protein-protein interactions
00:17:39.08 are very important.
00:17:41.17 We now know,
00:17:43.06 through work of Shiven Kapur
00:17:45.07 and others in my lab,
00:17:47.01 what the basis for this is.
00:17:49.11 So, it turns out that these carrier proteins,
00:17:51.29 which are helical proteins,
00:17:54.11 have different helices
00:17:56.10 -- different portions of this helical protein --
00:17:59.12 that dictate specificity at different events.
00:18:02.22 How do we know that?
00:18:05.02 So, you can take this red carrier protein
00:18:07.23 that talks to this red acceptor module,
00:18:12.06 replace it with the green carrier protein,
00:18:15.05 it doesn't do a good job of talking
00:18:17.14 to this acceptor module,
00:18:19.12 but now if you just put a portion of this helix
00:18:22.14 into this protein,
00:18:24.14 now this chimeric protein
00:18:26.19 is just as good,
00:18:28.12 or almost just as good,
00:18:30.06 as this red carrier protein
00:18:32.23 in delivering cargo
00:18:34.21 through this chain translocation event,
00:18:37.10 suggesting that it's this helix over here
00:18:41.02 that plays a very important role
00:18:43.08 in dictating the specificity
00:18:45.02 during chain translocation.
00:18:47.28 You can do exactly analogous experiments
00:18:50.05 in that other theoretical reaction,
00:18:53.22 the chain elongation reaction
00:18:55.23 where that carbon-carbon bond is formed
00:18:58.00 between the extender unit and the growing polyketide chain
00:19:01.23 to give you this newly elongated chain.
00:19:04.25 You can kinetically isolate this event,
00:19:07.10 you can use the same substrates,
00:19:10.20 same catalysts,
00:19:12.28 just change the identity of this carrier protein.
00:19:16.06 And what you see is,
00:19:17.28 depending on which module you use,
00:19:19.24 you can have big effects
00:19:21.23 on the specificity constant over here,
00:19:24.11 as Alice Chen showed in these experiments.
00:19:27.22 You can use the same kind of approaches
00:19:30.10 that Shiven Kapur used
00:19:32.05 to make chimeric carrier proteins
00:19:34.11 and show which portions
00:19:36.16 of these carrier proteins
00:19:38.12 play a critical role in dictating the specificity
00:19:42.22 that is measured in these parameters.
00:19:45.11 And so the take-home message
00:19:47.23 from experiments like this
00:19:51.03 is that even though chain elongation
00:19:55.07 and chain translocation
00:19:57.19 are sequential events
00:20:00.19 along the assembly line,
00:20:02.16 and even though all of those events
00:20:04.24 involve ketosynthase-acyl carrier protein interactions,
00:20:09.14 the specificity of those events
00:20:12.04 is being driven by different faces
00:20:14.23 at this carrier protein.
00:20:17.21 So this blue face of this carrier protein
00:20:20.26 is what's driving the translocation reaction,
00:20:24.17 whereas the yellow face of the carrier protein
00:20:27.19 is what's driving the elongation reaction.
00:20:31.20 So you have this Janus-like biochemistry
00:20:36.01 in these carrier proteins.
00:20:38.19 And the reason that allows you to do things
00:20:42.19 in a vectorial manner
00:20:45.13 is because now you can set up
00:20:48.18 so that a carrier protein
00:20:51.06 can only recognize the next module.
00:20:54.06 How do we know that?
00:20:56.01 We know that through experiments
00:20:57.23 where we engineer these carrier proteins
00:21:00.06 to be confused carrier proteins.
00:21:03.07 This is one example of that experiment,
00:21:05.16 where we engineer a module
00:21:07.29 that stutters.
00:21:10.09 So if you take a natural module,
00:21:12.12 like this one shown over here,
00:21:14.09 and its own carrier protein over here,
00:21:16.18 you give it a nucleophilic substrate
00:21:18.23 and electrophilic growing polyketide chain,
00:21:22.10 and you ask it to catalyze chain elongation,
00:21:25.00 it'll do so,
00:21:27.06 it'll do one round of chain elongation,
00:21:31.01 and it'll stop at this point.
00:21:34.07 And the reason it stops at this point
00:21:36.28 and doesn't go back
00:21:39.09 to the same carrier protein
00:21:41.08 is that this carrier protein
00:21:43.27 is a partner of this ketosynthase
00:21:46.17 in this chain elongation reaction
00:21:49.10 that forms this critical carbon-carbon bond,
00:21:52.22 but it is not a partner
00:21:55.16 for chain translocation.
00:21:57.16 Its chain translocation partner
00:21:59.13 is elsewhere on that assembly line.
00:22:02.14 And so the way you can test that
00:22:05.22 is you can take the recognition features
00:22:08.14 of the next carrier protein,
00:22:11.29 build them into this system
00:22:14.16 to generate a chimeric carrier protein
00:22:17.26 that can not only do a chain elongation event,
00:22:21.13 but this chimeric thing
00:22:23.12 can then donate it back
00:22:25.10 to this ketosynthase
00:22:27.19 and go through yet another round of chain elongation
00:22:30.11 to give you a longer module.
00:22:32.15 So here you've got a module
00:22:34.19 that is stuttering;
00:22:37.18 that's doing two rounds of elongation
00:22:40.07 instead of one and only one round,
00:22:42.10 because we've kind of scrambled up its recognition features.
00:22:47.02 And so what that allows me to do
00:22:49.10 is to leave you with a basic picture
00:22:52.17 of how you might wanna think about this assembly line.
00:22:56.12 So, I showed you this cartoon.
00:22:59.27 The question we're asking is,
00:23:01.29 how does the growing polyketide chain
00:23:05.10 pick one and only one path?
00:23:07.17 It's going through at least
00:23:09.17 300 Ångstroms of distance
00:23:11.19 as it travels through these modules
00:23:14.21 from one active site to another,
00:23:17.08 and comes out as a 6-Deoxyerythronolide B.
00:23:21.11 How does it pick one and only one path
00:23:24.08 to take through this entire assembly line?
00:23:27.27 And so, if we isolate just these two modules,
00:23:31.05 Modules 2 and 3,
00:23:33.24 I can show you what I think
00:23:36.05 might be a model for how this works.
00:23:39.18 So we start off
00:23:42.00 with the upstream module,
00:23:44.12 this pink module,
00:23:46.26 bound to its carrier protein
00:23:49.16 in the elongation state
00:23:51.19 -- what Jencks would call...
00:23:53.28 has specificity for the elongation state --
00:23:57.06 and that specificity
00:23:59.06 is derived from this yellow portion
00:24:01.07 of the carrier protein
00:24:03.11 that docks into this active site
00:24:05.14 and puts its substrate
00:24:07.17 where it belongs.
00:24:09.25 Meanwhile, the green carrier protein
00:24:11.24 of this green module
00:24:13.28 is just sitting in neutral mode.
00:24:16.25 So this carrier protein
00:24:19.02 then catalyzes chain elongation,
00:24:22.26 it yanks out that chain,
00:24:25.01 it can't return it back to the same site
00:24:27.05 because it doesn't have the appropriate recognition features,
00:24:30.27 instead its got recognition features, now,
00:24:34.00 using that panhandle linker,
00:24:36.06 that docking site that was identified as well as this helix
00:24:41.14 that Kapur identified.
00:24:44.02 It docks into this module,
00:24:46.11 but in a different orientation,
00:24:48.11 so now it can translocate
00:24:50.20 the chain to the next module.
00:24:54.14 When that happens,
00:24:56.13 that carrier protein goes back
00:24:58.13 into the neutral mode
00:25:00.15 and this carrier protein,
00:25:02.03 which is charged with the extender unit,
00:25:04.09 comes in to do the elongation reaction.
00:25:07.15 And so this three-stroke process
00:25:10.11 goes through again and again
00:25:12.19 to give you the final product,
00:25:15.19 to allow chains
00:25:17.13 to move forward through the assembly line.
00:25:21.10 And so I'll leave you with this picture.
00:25:24.19 I started with the picture to the left,
00:25:27.13 to motivate these remarkable antibiotic machines.
00:25:31.16 I've added one more picture
00:25:33.11 that I suspect most of you
00:25:34.26 who've had a class in biochemistry recognize.
00:25:38.10 It is the picture that we often use
00:25:40.18 to describe how proteins are made
00:25:43.06 on ribosomes.
00:25:47.24 Let me ask you the following question:
00:25:51.03 what do these pictures mean to you?
00:25:54.27 Chances are if I just flashed
00:25:57.11 both these pictures in front of you,
00:26:00.10 you would immediately recognize
00:26:02.11 what I'm saying.
00:26:04.27 If, on the other hand,
00:26:07.08 you had just arrived today from Mars,
00:26:10.08 and I showed you these pictures,
00:26:13.02 they would mean absolutely nothing to you.
00:26:16.29 So why do these pictures mean so much to you?
00:26:21.00 I would submit to you they mean a lot to you
00:26:24.03 because you have seen the movie in each case,
00:26:28.04 and that is where I will leave you
00:26:30.21 as the future of my field.
00:26:35.12 What I showed you in the past three slides
00:26:39.13 were essentially the first three frames
00:26:42.22 of a movie
00:26:45.15 that will be the DEBS movie
00:26:47.26 over the next decade.
00:26:50.09 There's no Hollywood,
00:26:51.26 no Disney as yet,
00:26:54.06 but it's backed up by
00:26:58.01 data based upon...
00:27:00.11 through a combination of biochemistry,
00:27:03.14 structural biology,
00:27:05.09 and similar types of chemical/biological approaches.
00:27:10.01 Our challenge for this field
00:27:11.27 is to take those static pictures
00:27:15.06 that I've been showing you so far
00:27:17.15 and convert them into a movie,
00:27:19.22 so once you've seen that movie,
00:27:21.29 you will recognize the engineering implications
00:27:25.01 of every state
00:27:27.07 in every one of those thousands of assembly lines
00:27:30.20 that nature has give you,
00:27:32.21 and be able to rationally think
00:27:34.23 about how to recombine them,
00:27:36.23 or how to extract interesting antibiotics
00:27:39.23 from the right kind of assembly line in nature.
00:27:43.06 I can't do that today,
00:27:46.17 nor can any of my coworkers.
00:27:49.24 But I hope this inspires you
00:27:52.16 to think about how that might be done in the future.
00:27:55.03 This brings me to the end of the lecture.
00:27:58.16 I'd like to thank you for your attention.
00:28:02.24 I'd like to thank all of my students
00:28:05.16 and postdoctoral fellows
00:28:07.21 who, over the years,
00:28:09.16 have worked with me on DEBS
00:28:11.26 and related polyketide assembly lines.
00:28:14.29 Their names show up to the left of the slide,
00:28:18.27 and I've been acknowledging their papers
00:28:21.18 as we've gone along through
00:28:23.20 these different modules.
00:28:26.27 And I'd like to also thank my collaborators:
00:28:30.20 David Cane at Brown University,
00:28:33.05 with whom I've had a long-standing collaboration
00:28:36.19 on the study of DEBS,
00:28:38.23 as well as my collaborators
00:28:41.00 at the synchrotron
00:28:42.23 -- SLAC synchrotron --
00:28:44.21 Matthews Irimpan,
00:28:46.02 Tsutomu Matsui,
00:28:47.25 and Thomas Weiss,
00:28:49.11 with whom we've had very productive collaborations
00:28:51.25 associated with the
00:28:54.05 crystallographic and SAXS characterization
00:28:56.29 of DEBS.
00:28:58.12 And last, but not least,
00:29:00.03 I'd like to thank the NIH
00:29:02.01 for having supported all our work on DEBS
00:29:05.13 over the past 15 years.
00:29:07.27 Thank you.
- Can you think of an interesting experiment involving polyketide assembly pathways that was not presented in the lectures?
- Did this lecture leave you with any unanswered questions?
- What do you think is a major hurdle to engineering pathways like this to create a non-natural product?
Paper for this Session’s Discussion
Walker MC, Thuronyi BW, Charkoudian LK, Lowry B, Khosla C, Chang MC.Expanding the fluorine chemistry of living systems using engineered polyketide synthase pathways. Science. 2013. 341: 1089-1094. PMID: 24009388
Discussion Questions for the Paper
- What was the overall goal of this research? Why would achieving this goal be useful?
- How are the experiments shown in figures 2A and 2B similar? What is different about them
- Describe the experimental set-up depicted in Figure 3. What are the two main results from this experiment?
- What is the purpose of making the different mutations shown in Figures 4C and D? How do these mutations affect the DEBS modules, and how is this useful?
- What traits of E. coli are important for the in vivo chain extension reactions using fluoromalonyl-CoA?
Chaitan Khosla is Professor of Chemical Engineering, Chemistry and Biochemistry at Stanford University. Khosla received his B. Tech from the Indian Institute of Technology, his Ph.D. from the California Institute of Technology and he was a post-doctoral fellow at the John Innes Centre in the U.K. Khosla’s research interests bring together the fields of chemistry… Continue Reading