An Introduction to Polyketide Assembly Lines

Transcript of Part 1: An Introduction to Polyketide Assembly Lines

00:00:07.09	Greetings.
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
00:04:19.29	Erythromycin.
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,
00:17:43.17	DEBS,
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.