Session 4: 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.