WEBVTT 00:00:01.270 --> 00:00:03.700 The following content is provided under a Creative 00:00:03.700 --> 00:00:05.240 Commons license. 00:00:05.240 --> 00:00:07.540 Your support will help MIT OpenCourseWare 00:00:07.540 --> 00:00:11.900 continue to offer high-quality educational resources for free. 00:00:11.900 --> 00:00:14.530 To make a donation or view additional materials 00:00:14.530 --> 00:00:18.470 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:18.470 --> 00:00:19.660 at ocw.mit.edu. 00:00:26.630 --> 00:00:30.020 ELIZABETH NOLAN: We're going to get started 00:00:30.020 --> 00:00:33.200 and what we'll do today is continue 00:00:33.200 --> 00:00:35.180 with fatty acid synthase. 00:00:35.180 --> 00:00:39.350 Because that's the paradigm for these macromolecular machines, 00:00:39.350 --> 00:00:42.410 like the PKS, and then we'll go over 00:00:42.410 --> 00:00:44.630 the logic of polyketide synthases. 00:00:44.630 --> 00:00:50.120 So we left off last time with this discussion 00:00:50.120 --> 00:00:52.940 about some molecules that will be involved 00:00:52.940 --> 00:00:55.880 and in particular thioesters, and I 00:00:55.880 --> 00:01:01.160 asked about the alpha H. So just going back 00:01:01.160 --> 00:01:03.630 to introductory organic chemistry, what 00:01:03.630 --> 00:01:09.032 are the properties of this atom here? 00:01:09.032 --> 00:01:10.240 AUDIENCE: [INAUDIBLE] acidic. 00:01:10.240 --> 00:01:11.157 ELIZABETH NOLAN: Yeah. 00:01:11.157 --> 00:01:11.660 OK, right. 00:01:11.660 --> 00:01:13.190 So this is acidic. 00:01:13.190 --> 00:01:14.255 So if you have-- 00:01:24.170 --> 00:01:24.670 OK? 00:01:24.670 --> 00:01:28.120 So what that means is if there is a base that can deprotonate 00:01:28.120 --> 00:01:30.240 that, we can get an enolate. 00:01:30.240 --> 00:01:31.840 OK, and this is the type of chemistry 00:01:31.840 --> 00:01:35.170 that's going to be happening with the thioesters that 00:01:35.170 --> 00:01:40.060 are used in fatty acid synthase and also polyketide synthase. 00:01:40.060 --> 00:01:42.490 And just to rewind a little bit more, 00:01:42.490 --> 00:01:45.730 if we think about carbon-carbon bond forming reactions 00:01:45.730 --> 00:01:48.790 in nature, which is what's happening in fatty acid 00:01:48.790 --> 00:01:51.580 biosynthesis and in polyketide biosynthesis, 00:01:51.580 --> 00:01:55.690 effectively, nature uses three different types of reaction. 00:01:55.690 --> 00:01:58.720 OK, so one is the aldol, two are the Claisen, 00:01:58.720 --> 00:02:00.230 and three [INAUDIBLE] transfer. 00:02:00.230 --> 00:02:04.420 OK, and so we're going to see Claisen condensations in FAS 00:02:04.420 --> 00:02:06.820 and PKS biosynthesis. 00:02:06.820 --> 00:02:09.130 And then after spring break, when 00:02:09.130 --> 00:02:14.123 Joanne starts with cholesterol biosynthesis, 00:02:14.123 --> 00:02:15.790 that will involve [INAUDIBLE] transfers. 00:02:15.790 --> 00:02:19.030 And hopefully, you've seen aldol reactions sometime 00:02:19.030 --> 00:02:21.640 before within biochemistry here. 00:02:21.640 --> 00:02:22.330 OK? 00:02:22.330 --> 00:02:25.630 So we need to think about just what the general Claisen 00:02:25.630 --> 00:02:27.910 condensation is that we're going to be seeing here 00:02:27.910 --> 00:02:30.010 and the consequences of this acidic proton. 00:02:33.160 --> 00:02:36.160 So also just keep in mind, rewinding a little more, 00:02:36.160 --> 00:02:40.350 nature uses thioesters not esters, 00:02:40.350 --> 00:02:43.650 and so the alpha H is more acidic. 00:02:43.650 --> 00:02:46.780 The carbonyl is more activated for nucloephilic attack. 00:02:46.780 --> 00:02:48.720 And there's some resonance arguments 00:02:48.720 --> 00:02:51.880 and orbital overlap arguments that 00:02:51.880 --> 00:02:56.320 can guide those conclusions, if you wish to do them here. 00:02:56.320 --> 00:02:56.820 OK. 00:02:56.820 --> 00:03:03.983 So let's imagine that we have a thioester. 00:03:09.540 --> 00:03:10.515 We have a base. 00:03:15.510 --> 00:03:22.820 OK, that's going to be [INAUDIBLE],, which 00:03:22.820 --> 00:03:24.240 is going to get us to here. 00:03:33.840 --> 00:03:41.886 So this is our nucleophile, and what you'll see coming forward 00:03:41.886 --> 00:03:43.870 is an enolate. 00:03:43.870 --> 00:03:50.188 So imagine we have that, and we add it with another thioester, 00:03:50.188 --> 00:03:51.990 and here's our electrophile. 00:03:56.760 --> 00:03:58.320 What do we get? 00:04:09.320 --> 00:04:23.250 We get formation of a beta-keto thioester, which is the Claisen 00:04:23.250 --> 00:04:24.440 condensation product. 00:04:34.903 --> 00:04:37.383 OK, you have two thioesters. 00:04:40.221 --> 00:04:41.170 OK? 00:04:41.170 --> 00:04:45.220 So effectively, this acyl thioester is doubly activated, 00:04:45.220 --> 00:04:46.630 so it can be-- 00:04:46.630 --> 00:04:47.790 did I lose it? 00:04:47.790 --> 00:04:51.360 Oh no, problems. 00:04:51.360 --> 00:04:52.990 Sorry about that. 00:04:52.990 --> 00:04:57.810 It can be activated as an electrophile at the C1 00:04:57.810 --> 00:05:00.470 position, so next door to the sulfur. 00:05:00.470 --> 00:05:04.090 And it can be activated as a nucleophile at the C2 position 00:05:04.090 --> 00:05:04.750 here. 00:05:04.750 --> 00:05:06.730 So this is the general chemistry that's 00:05:06.730 --> 00:05:09.970 going to be happening by FAS and PKS 00:05:09.970 --> 00:05:12.640 in terms of forming carbon-carbon bonds 00:05:12.640 --> 00:05:15.650 between monomers here. 00:05:15.650 --> 00:05:16.150 OK? 00:05:16.150 --> 00:05:21.180 So in fatty acid synthase, we have two monomer units. 00:05:31.730 --> 00:05:32.680 OK? 00:05:32.680 --> 00:05:36.400 So we have acetyl-CoA and malonyl-CoA. 00:05:49.530 --> 00:06:01.700 Acetyl-CoA is the starter unit, sometimes called unit 0, 00:06:01.700 --> 00:06:05.465 and then malonyl-CoA is the extender. 00:06:28.340 --> 00:06:32.220 And so recall that in fatty acid biosynthesis, 00:06:32.220 --> 00:06:36.690 each elongation event adds two carbons, 00:06:36.690 --> 00:06:39.640 and if we look at malonyl-CoA, we have three here. 00:06:39.640 --> 00:06:40.140 Right? 00:06:40.140 --> 00:06:43.470 So there's decarboxylation of malonyl-CoA 00:06:43.470 --> 00:06:55.870 to generate a C2 unit, and there's 00:06:55.870 --> 00:06:58.010 details of that in the lecture 15 notes. 00:07:07.450 --> 00:07:19.990 And SCoA is coenzyme A, here, and there's some information 00:07:19.990 --> 00:07:23.140 as to the biosynthesis of these starter and extender units 00:07:23.140 --> 00:07:23.740 in the notes. 00:07:23.740 --> 00:07:26.840 We're not going to go over that in lecture here. 00:07:26.840 --> 00:07:32.710 So in terms of using these monomers to obtain 00:07:32.710 --> 00:07:35.620 fatty acids, first what we're going to go over 00:07:35.620 --> 00:07:39.390 are the domains in FAS. 00:07:39.390 --> 00:07:41.590 And so we can consider domains that 00:07:41.590 --> 00:07:45.880 are required for extension of the fatty acid chain 00:07:45.880 --> 00:07:47.590 and then domains that are required 00:07:47.590 --> 00:07:52.680 for tailoring of that effectively to reduce 00:07:52.680 --> 00:07:57.100 the carbonyl, as shown. 00:07:57.100 --> 00:07:58.600 And we're going to go through these, 00:07:58.600 --> 00:08:00.058 because what we're going to find is 00:08:00.058 --> 00:08:03.670 that with polyketide biosynthesis, 00:08:03.670 --> 00:08:06.170 the same types of domains are used. 00:08:06.170 --> 00:08:08.790 So this logic extends there. 00:08:08.790 --> 00:08:09.290 OK. 00:08:09.290 --> 00:08:18.730 So first, we have domains required 00:08:18.730 --> 00:08:36.539 for elongation of the fatty acid chain by one two-carbon unit. 00:08:39.669 --> 00:08:40.169 OK. 00:08:40.169 --> 00:08:44.430 So these include domains that may be abbreviated 00:08:44.430 --> 00:08:58.430 as AAT or MAT, and they can be grouped as AT 00:08:58.430 --> 00:09:05.955 and stand for acetyl or malonyltransferase. 00:09:15.000 --> 00:09:15.540 OK. 00:09:15.540 --> 00:09:30.370 We have an Acyl Carrier Protein, ACP, 00:09:30.370 --> 00:09:41.390 and this carries the growing chain between the domains 00:09:41.390 --> 00:09:43.590 of fatty acid synthase. 00:09:43.590 --> 00:09:45.690 And so in recitation this week, you're 00:09:45.690 --> 00:09:49.530 going to see how these domains move around 00:09:49.530 --> 00:09:54.000 and talk about the length of this acyl carrier protein. 00:09:54.000 --> 00:09:55.830 We also have the ketosynthase. 00:10:03.450 --> 00:10:06.540 So what the ketosynthase does is it accepts the growing 00:10:06.540 --> 00:10:18.460 chain from the acyl carrier protein, 00:10:18.460 --> 00:10:31.818 and it catalyzes the Claisen condensation 00:10:31.818 --> 00:10:32.735 with the next monomer. 00:10:41.660 --> 00:10:43.790 And what we'll see is that this ketosynthase 00:10:43.790 --> 00:11:01.000 uses covalent catalysis, and via a cysteine thiolate residue. 00:11:04.440 --> 00:11:07.430 So these are the key domains required 00:11:07.430 --> 00:11:09.560 for elongation of the chain. 00:11:09.560 --> 00:11:10.060 OK? 00:11:10.060 --> 00:11:17.240 And then what we also need are domains required for tailoring, 00:11:17.240 --> 00:11:22.460 and just to clarify, I'm defining domain here 00:11:22.460 --> 00:11:27.800 as a polypeptide with a single enzymatic activity. 00:11:27.800 --> 00:11:30.650 So domains can be connected to one another, 00:11:30.650 --> 00:11:35.030 or they can be standalone in different types of synthases, 00:11:35.030 --> 00:11:37.250 but domain means polypeptide with 00:11:37.250 --> 00:11:38.930 a single enzymatic activity. 00:11:46.880 --> 00:11:49.450 So what are the domains required for tailoring? 00:11:54.250 --> 00:12:03.520 And these work after addition of the C2 unit 00:12:03.520 --> 00:12:05.630 to the growing chain. 00:12:05.630 --> 00:12:07.850 So first, there's a ketoreductase. 00:12:18.370 --> 00:12:20.960 And as indicated, what this enzyme does 00:12:20.960 --> 00:12:40.090 is it reduces the carbonyl of the previous unit to an OH 00:12:40.090 --> 00:12:48.100 and uses an NADPH H plus. 00:12:48.100 --> 00:12:54.250 We also have the dehydratase here, 00:12:54.250 --> 00:13:00.730 and this forms an alpha, beta-alkene from the product 00:13:00.730 --> 00:13:03.730 of the ketoreductase action. 00:13:03.730 --> 00:13:07.240 And then we have an enoyl reductase 00:13:07.240 --> 00:13:18.030 that reduces this alpha, beta-alkene, 00:13:18.030 --> 00:13:24.670 and this also requires NADPH H plus here. 00:13:24.670 --> 00:13:28.150 And then some fatty synthases use 00:13:28.150 --> 00:13:32.020 a domain called a thioesterase for chain release, 00:13:32.020 --> 00:13:35.210 and that's noted as TE. 00:13:43.930 --> 00:13:49.820 And we'll see thioesterases in the PKS and in our PS sections 00:13:49.820 --> 00:13:50.320 here. 00:13:52.840 --> 00:13:57.130 So one comment regarding the acyl carrier protein, 00:13:57.130 --> 00:14:00.490 and then we'll just look at the fatty acid synthase cycle 00:14:00.490 --> 00:14:03.190 and see how these domains are acting. 00:14:22.090 --> 00:14:27.010 So in order for the acyl carrier protein 00:14:27.010 --> 00:14:29.530 to carry this growing chain, it first 00:14:29.530 --> 00:14:32.470 needs to be post-translationally modified 00:14:32.470 --> 00:14:34.600 with what's called a PPant arm. 00:14:34.600 --> 00:14:38.200 And that arm provides the ability 00:14:38.200 --> 00:14:43.240 to have these monomers, or growing chains, linked 00:14:43.240 --> 00:14:46.660 via a thioester. 00:14:46.660 --> 00:14:50.080 And so just to go over this post-translational 00:14:50.080 --> 00:14:56.550 modification, so post-translational modification 00:14:56.550 --> 00:15:01.690 of acyl carrier protein with the PPant arm. 00:15:05.150 --> 00:15:05.650 OK. 00:15:05.650 --> 00:15:13.520 If we consider apo acyl carrier protein, 00:15:13.520 --> 00:15:16.420 and apo means that the PPant arm is not attached. 00:15:21.720 --> 00:15:22.890 There's a serine residue. 00:15:27.030 --> 00:15:35.610 An enzyme called the PPTase comes along, 00:15:35.610 --> 00:15:38.070 and it allows for post-translational modification 00:15:38.070 --> 00:15:49.410 of this serine using CoASH, releasing 3', 5'-ADP to give 00:15:49.410 --> 00:15:55.970 ACP post-translationally modified with the PPant arm. 00:15:55.970 --> 00:15:56.470 OK? 00:15:56.470 --> 00:15:58.012 And we'll look at the actual chemical 00:15:58.012 --> 00:16:00.270 structures in a minute. 00:16:00.270 --> 00:16:03.780 What I want to point out is that throughout this unit, 00:16:03.780 --> 00:16:07.920 this squiggle, some form of squiggle here, 00:16:07.920 --> 00:16:10.320 is the abbreviation for the PPant arm. 00:16:18.760 --> 00:16:19.770 OK? 00:16:19.770 --> 00:16:28.230 And this is very flexible and about 20 angstroms in length. 00:16:35.130 --> 00:16:38.850 So what does this actually look like? 00:16:38.850 --> 00:16:41.420 So here we have CoASH. 00:16:44.090 --> 00:16:48.840 So PPant is an abbreviation for phosphopantetheine, here, 00:16:48.840 --> 00:16:53.220 this moiety, and here's the 3', 5'-ADP. 00:16:53.220 --> 00:16:58.410 And so effectively, what's shown on the board is repeated here. 00:16:58.410 --> 00:17:01.050 Except for here, we're seeing the full structure 00:17:01.050 --> 00:17:04.650 of the phosphopantetheinylated acyl carrier protein. 00:17:04.650 --> 00:17:07.560 So this squiggle abbreviation indicates 00:17:07.560 --> 00:17:10.050 this post-translational modification 00:17:10.050 --> 00:17:11.999 onto a serine residue of the ACP. 00:17:14.760 --> 00:17:17.060 Just as an example of structure, so here 00:17:17.060 --> 00:17:21.000 is a structure of acyl carrier protein from E. coli. 00:17:21.000 --> 00:17:24.060 It's about 10 kilodaltons, so not very big, 00:17:24.060 --> 00:17:28.010 and we see the PPant arm here attached. 00:17:28.010 --> 00:17:30.330 OK? 00:17:30.330 --> 00:17:40.670 So if we think about fatty acids biosynthesis, 00:17:40.670 --> 00:17:46.610 we can think about this in three steps, better iterated. 00:17:49.410 --> 00:17:49.910 OK. 00:17:49.910 --> 00:17:56.810 So first we have loading, so the acyl carrier proteins need 00:17:56.810 --> 00:17:59.330 to be loaded with monomers. 00:17:59.330 --> 00:18:03.560 Sometimes, this step the reactions 00:18:03.560 --> 00:18:05.810 are described as priming reactions. 00:18:12.440 --> 00:18:18.690 We have initiation and elongation 00:18:18.690 --> 00:18:24.930 all grouped together here and, three, at some point, 00:18:24.930 --> 00:18:25.860 a termination. 00:18:28.920 --> 00:18:29.740 OK? 00:18:29.740 --> 00:18:32.440 So we've thought about these before from the standpoint 00:18:32.440 --> 00:18:35.500 of biological polymerizations. 00:18:42.650 --> 00:18:46.640 So what about the FAS cycle? 00:18:46.640 --> 00:18:51.350 Here's one depiction, and I've provided multiple depictions 00:18:51.350 --> 00:18:52.730 in the lecture 15 notes. 00:18:52.730 --> 00:18:55.940 Because some people find different cycles easier 00:18:55.940 --> 00:18:58.610 than others, but let's just take a look. 00:18:58.610 --> 00:19:03.110 So this charts out the various domains-- 00:19:03.110 --> 00:19:06.500 the starter and the extender and then the chemistry that 00:19:06.500 --> 00:19:08.930 occurs on these steps. 00:19:08.930 --> 00:19:12.770 And so what needs to happen is that there 00:19:12.770 --> 00:19:16.820 needs to be some loading and initiation where 00:19:16.820 --> 00:19:22.370 the acetyl-CoA is loaded onto an acyl carrier protein. 00:19:22.370 --> 00:19:27.530 So that's shown here via transferase here, 00:19:27.530 --> 00:19:30.230 and then, from the acyl carrier protein, 00:19:30.230 --> 00:19:34.340 this monomer is loaded onto the ketosynthase. 00:19:34.340 --> 00:19:36.950 If we look here, we have one of our extender units, 00:19:36.950 --> 00:19:40.250 the malonyl-CoA, and the CO2 unit 00:19:40.250 --> 00:19:42.610 that gets removed during decarboxylation, 00:19:42.610 --> 00:19:44.430 as shown in this light blue. 00:19:44.430 --> 00:19:44.930 OK? 00:19:44.930 --> 00:19:49.580 We need to have this extender unit also transferred 00:19:49.580 --> 00:19:53.090 to an acyl carrier protein via the action of an AT. 00:19:53.090 --> 00:19:54.800 So we see lots of the CoA. 00:19:54.800 --> 00:19:58.040 Here we have the acyl carrier protein with the PPant arm. 00:19:58.040 --> 00:19:59.210 It's not a squiggle here. 00:19:59.210 --> 00:20:03.110 It is the next one with this malonyl unit loaded. 00:20:03.110 --> 00:20:07.490 There's a decarboxylation, and what do we see happening here? 00:20:07.490 --> 00:20:10.370 We have a chain elongation event, 00:20:10.370 --> 00:20:12.950 so Claisen condensation catalyzed 00:20:12.950 --> 00:20:16.760 by the ketosynthase between the starter and the first extender 00:20:16.760 --> 00:20:19.280 to give us this beta-keto thioester. 00:20:19.280 --> 00:20:21.800 So once this carbon-carbon bond is formed to give us 00:20:21.800 --> 00:20:26.000 the beta-keto thioester, there's processing of the beta carbon 00:20:26.000 --> 00:20:27.530 via those tailoring domains-- 00:20:30.240 --> 00:20:33.050 the dehydratase and the enoyl reductase. 00:20:33.050 --> 00:20:38.210 And so we see reduction of the beta ketone here, 00:20:38.210 --> 00:20:41.630 we see formation of the alkene, and then we see reduction 00:20:41.630 --> 00:20:43.220 to get us to this point. 00:20:43.220 --> 00:20:47.720 And so this cycle can repeat itself until, at some point, 00:20:47.720 --> 00:20:49.040 there's a termination event. 00:20:49.040 --> 00:20:52.850 And in this case here, we see a thioesterase catalyzing 00:20:52.850 --> 00:20:57.260 hydrolytic release of the fatty acid chain. 00:20:57.260 --> 00:20:59.930 This is the depiction you'll see in recitation today, 00:20:59.930 --> 00:21:01.340 or saw before. 00:21:01.340 --> 00:21:03.710 And I guess what I like about this depiction is 00:21:03.710 --> 00:21:07.070 that you see color coding separating 00:21:07.070 --> 00:21:09.740 the elongation and the domains involved 00:21:09.740 --> 00:21:14.330 in elongation with then the processing of the beta ketone 00:21:14.330 --> 00:21:17.210 here and then termination. 00:21:17.210 --> 00:21:18.220 OK. 00:21:18.220 --> 00:21:23.170 So we get some fatty acid from this. 00:21:23.170 --> 00:21:27.610 And so where we're going to go with this overview is looking 00:21:27.610 --> 00:21:32.770 at the polyketides and to ask what similar and different 00:21:32.770 --> 00:21:36.700 in terms of polyketide biosynthesis? 00:21:36.700 --> 00:21:39.400 And so where we can begin with thinking 00:21:39.400 --> 00:21:44.770 about that is asking what are the starters and extenders? 00:21:44.770 --> 00:21:47.620 And so these are the starters and extenders 00:21:47.620 --> 00:21:50.020 we saw for fatty acids, and here are 00:21:50.020 --> 00:21:54.290 the starters and extenders for polyketides, so very similar. 00:21:54.290 --> 00:21:54.790 Right? 00:21:54.790 --> 00:21:57.010 We just see that there's some additional options, 00:21:57.010 --> 00:21:59.630 so we also have this propionyl-CoA here. 00:21:59.630 --> 00:22:02.500 In addition to malonyl-CoA as an extender, 00:22:02.500 --> 00:22:07.880 we see that methylmalonyl-CoA can be employed. 00:22:07.880 --> 00:22:12.170 So what are the core domains of the PKS? 00:22:12.170 --> 00:22:16.640 They're similar to those of FAS, and we'll just focus 00:22:16.640 --> 00:22:19.020 on the PKS side of this table. 00:22:19.020 --> 00:22:21.950 So this is a helpful table when reviewing 00:22:21.950 --> 00:22:24.230 both types of assembly lines. 00:22:24.230 --> 00:22:27.320 So the core means that every module, 00:22:27.320 --> 00:22:31.310 which I'll define in a moment, contains these domains. 00:22:31.310 --> 00:22:33.860 So we see that there's a ketosynthase, 00:22:33.860 --> 00:22:37.280 an acyltransferase, and a thiolation domain. 00:22:37.280 --> 00:22:39.770 So this thiolation domain is the same 00:22:39.770 --> 00:22:42.020 as the acyl carrier protein. 00:22:42.020 --> 00:22:45.590 So there's different terminology used, and within the notes, 00:22:45.590 --> 00:22:49.220 I have some pages that are dedicated 00:22:49.220 --> 00:22:52.440 to these terminologies. 00:22:52.440 --> 00:22:54.250 OK? 00:22:54.250 --> 00:23:03.680 So for PKS, here, we have the ketosynthase, 00:23:03.680 --> 00:23:07.610 we have acetyltransferase, and then 00:23:07.610 --> 00:23:12.740 we have this T domain which equals acyl carrier 00:23:12.740 --> 00:23:14.340 protein here. 00:23:14.340 --> 00:23:15.530 OK? 00:23:15.530 --> 00:23:17.480 So then what about these tailoring 00:23:17.480 --> 00:23:20.930 domains that were required to produce the fatty acid? 00:23:20.930 --> 00:23:24.980 What we see in polyketide biosynthesis 00:23:24.980 --> 00:23:26.570 is that those domains are optional. 00:23:30.620 --> 00:23:44.670 So one or more of these domains may be in a given module. 00:24:02.870 --> 00:24:06.170 So that's an overview, and then we'll 00:24:06.170 --> 00:24:12.020 look at an example of some domains and modules. 00:24:12.020 --> 00:24:17.300 So we're going to focus on type 1 polyketide synthases. 00:24:17.300 --> 00:24:20.300 And in these, what we're going to see 00:24:20.300 --> 00:24:25.880 is that catalytic and carrier protein domains are fused, 00:24:25.880 --> 00:24:29.750 and they're organized into what we'll term modules. 00:24:29.750 --> 00:24:35.240 So a module is defined as a group of domains that's 00:24:35.240 --> 00:24:42.000 responsible for activating, forming the carbon-carbon bonds 00:24:42.000 --> 00:24:43.640 and tailoring a monomer. 00:24:43.640 --> 00:24:47.780 So there is an individual module for every monomer 00:24:47.780 --> 00:24:49.670 within the growing chain. 00:24:49.670 --> 00:24:54.200 And the order of the modules in the polyketide synthase 00:24:54.200 --> 00:24:56.720 determines the functional group status, 00:24:56.720 --> 00:25:00.020 and that functional group status is determined by whether or not 00:25:00.020 --> 00:25:01.940 these optional domains are there. 00:25:01.940 --> 00:25:04.590 OK? 00:25:04.590 --> 00:25:07.410 How do we look for modules? 00:25:07.410 --> 00:25:11.730 The easiest way is to look for one of these thiolation or ACP 00:25:11.730 --> 00:25:12.930 domains. 00:25:12.930 --> 00:25:15.150 So each module has one of these. 00:25:15.150 --> 00:25:18.180 So you can count your number of T domains, 00:25:18.180 --> 00:25:21.180 and then you know, OK, there's 7T domains, 00:25:21.180 --> 00:25:25.020 so there's 7 monomers, for instance. 00:25:25.020 --> 00:25:28.890 So each Claisen condensation is a chain elongation and chain 00:25:28.890 --> 00:25:32.340 translocation event. 00:25:32.340 --> 00:25:34.260 Keep in mind, the starting monomer-- 00:25:34.260 --> 00:25:37.650 so whether that's acetyl-CoA or propionyl-CoA-- 00:25:37.650 --> 00:25:39.780 does not contain a CO2 group. 00:25:39.780 --> 00:25:42.810 So there's no decarboxylation of the starting monomer, 00:25:42.810 --> 00:25:45.270 but decarboxylation of malonyl-CoA 00:25:45.270 --> 00:25:47.220 occurs, like in fatty acid synthase, 00:25:47.220 --> 00:25:50.670 and if that's the case, it provides a C2 unit. 00:25:50.670 --> 00:25:53.160 And if methylmalonyl-CoA is the extender, 00:25:53.160 --> 00:25:55.620 this decarboxylation provides a C3 unit 00:25:55.620 --> 00:25:58.470 because of that methyl group. 00:25:58.470 --> 00:26:01.680 So key difference, as we just saw, 00:26:01.680 --> 00:26:05.160 in fatty acid biosynthesis, we have complete reduction of that 00:26:05.160 --> 00:26:08.040 beta-keto group in every elongation cycle 00:26:08.040 --> 00:26:10.290 because of these three tailoring domains-- 00:26:10.290 --> 00:26:12.820 the KR, DH, and ER. 00:26:12.820 --> 00:26:16.260 In PKS, what can happen is that reduction 00:26:16.260 --> 00:26:19.440 of this beta-keto group may not happen at all, 00:26:19.440 --> 00:26:23.280 or it may be incomplete in each elongation step. 00:26:23.280 --> 00:26:25.350 So what that means is that polyketides 00:26:25.350 --> 00:26:29.670 retain functional groups during chain elongation. 00:26:29.670 --> 00:26:32.160 And if you look back at some of the structures that 00:26:32.160 --> 00:26:34.710 were in the notes from last time, 00:26:34.710 --> 00:26:37.110 you'll see that, in terms of ketones, hydroxyls, 00:26:37.110 --> 00:26:39.450 double bonds, et cetera. 00:26:39.450 --> 00:26:41.130 And also, the other point to note 00:26:41.130 --> 00:26:43.770 is that there can be additional chemistry, 00:26:43.770 --> 00:26:48.580 and that these assembly lines where polyketide synthases, 00:26:48.580 --> 00:26:51.480 non-ribosomal peptide synthatases can contain what 00:26:51.480 --> 00:26:53.340 are called optional domains. 00:26:53.340 --> 00:26:54.960 So these are additional domains that 00:26:54.960 --> 00:26:58.020 are not required for formation of the carbon-carbon bond 00:26:58.020 --> 00:27:01.380 or amide bond in non-ribosomal peptide synthases. 00:27:01.380 --> 00:27:03.630 But they can do other chemistry there, so 00:27:03.630 --> 00:27:07.140 imagine a methyltransferase, for instance, or some cyclization 00:27:07.140 --> 00:27:08.920 domain. 00:27:08.920 --> 00:27:19.675 So how do we show these domains and modules? 00:27:22.460 --> 00:27:29.990 So typically, a given synthase is depicted from left to right 00:27:29.990 --> 00:27:38.520 in order of domain and bond-forming reactions here. 00:27:38.520 --> 00:27:39.790 So let's just take a look. 00:27:42.930 --> 00:27:58.030 So if we consider PKS domains and modules, 00:27:58.030 --> 00:28:26.610 we're just going to look at a pretend assembly line. 00:28:26.610 --> 00:28:27.200 OK? 00:28:27.200 --> 00:28:35.560 So this I'm defining here as an optional domain. 00:28:42.830 --> 00:28:46.540 So in this depiction, going from left to right, 00:28:46.540 --> 00:28:54.130 each one of these circles is a domain, so 00:28:54.130 --> 00:28:59.230 a polypeptide with a single enzymatic activity. 00:28:59.230 --> 00:29:02.560 Note that they're all basically touching one 00:29:02.560 --> 00:29:07.480 another which indicates in these types of notations 00:29:07.480 --> 00:29:09.425 that the polypeptide continues. 00:29:09.425 --> 00:29:11.050 It's not two different proteins, but we 00:29:11.050 --> 00:29:14.140 have one polypeptide here. 00:29:14.140 --> 00:29:17.440 I said that there's modules, and we can identify modules 00:29:17.440 --> 00:29:19.760 by counting T domains. 00:29:19.760 --> 00:29:23.320 So here, we have three T domains. 00:29:23.320 --> 00:29:25.015 So effectively there's three modules. 00:29:27.960 --> 00:29:40.050 So we have a module here, we have a module here, 00:29:40.050 --> 00:29:41.560 and we have a module here. 00:29:47.740 --> 00:29:48.390 What do we see? 00:29:48.390 --> 00:29:52.170 Two of these modules have a ketosynthase, 00:29:52.170 --> 00:29:54.690 so that's the domain that catalyzes the Claisen 00:29:54.690 --> 00:29:56.490 condensation. 00:29:56.490 --> 00:30:01.360 We have no ketosynthase here, in this first module. 00:30:01.360 --> 00:30:02.370 Why is that? 00:30:02.370 --> 00:30:04.330 We're all the way to the left. 00:30:04.330 --> 00:30:08.440 This is effectively our starter or loading module. 00:30:08.440 --> 00:30:12.870 So the propionyl-CoA or acetyl-CoA 00:30:12.870 --> 00:30:15.450 will be here, as we'll see, and there's nothing 00:30:15.450 --> 00:30:19.000 upstream to catalyze a condensate event with. 00:30:19.000 --> 00:30:22.290 So there's no KS domain in the starting 00:30:22.290 --> 00:30:24.700 module here or loading module. 00:30:24.700 --> 00:30:25.200 OK. 00:30:25.200 --> 00:30:33.245 So this is often called loading or starter. 00:30:38.790 --> 00:30:45.030 So if we think about these optional domains for a minute 00:30:45.030 --> 00:30:46.620 and think about how they work. 00:30:50.730 --> 00:31:01.660 If we go back to fatty acid synthase, and let's 00:31:01.660 --> 00:31:06.040 just imagine we have this species attached. 00:31:06.040 --> 00:31:12.890 We have the action of the KR, the dehydratase, 00:31:12.890 --> 00:31:20.830 and the ER to give us the fully-reduced species. 00:31:20.830 --> 00:31:24.970 Where here, we have a CH2 to group 00:31:24.970 --> 00:31:27.070 rather than the beta-ketone. 00:31:32.350 --> 00:31:36.760 So what happens in PKS in terms of 00:31:36.760 --> 00:31:38.620 the different optional domains? 00:31:38.620 --> 00:31:41.650 So we could have this and have full reduction. 00:31:41.650 --> 00:31:47.990 We can imagine maybe there's no enoyl reductase. 00:31:47.990 --> 00:31:50.800 So the module has the ketoreductase 00:31:50.800 --> 00:31:55.390 and the dehydratase but no enoyl reductase, and so as a result, 00:31:55.390 --> 00:32:02.200 this polyketide ends up with a double bond here. 00:32:02.200 --> 00:32:03.540 OK? 00:32:03.540 --> 00:32:11.460 What if we have nobody dehydratase, like this? 00:32:11.460 --> 00:32:11.960 OK. 00:32:11.960 --> 00:32:14.855 We just work backwards from the FAS cycle. 00:32:18.340 --> 00:32:21.620 We'd be left with this OH group at the beta position. 00:32:21.620 --> 00:32:22.120 Right? 00:32:22.120 --> 00:32:28.580 And if we have none of them, so no ketoreductase, dehydratase, 00:32:28.580 --> 00:32:35.740 or enoyl reductase, the beta-ketone 00:32:35.740 --> 00:32:40.120 will be retained, here. 00:32:44.130 --> 00:32:47.520 So what this also means is that you can just 00:32:47.520 --> 00:32:57.110 look at some polyketide and assess 00:32:57.110 --> 00:33:00.080 what the situation is from the standpoint 00:33:00.080 --> 00:33:02.360 of these optional domains. 00:33:02.360 --> 00:33:05.370 So let's just take an example. 00:33:05.370 --> 00:33:13.960 If we have three cycles of elongation, and let's 00:33:13.960 --> 00:33:25.720 imagine we had an acetyl-CoA starter plus three malonyl-CoA. 00:33:30.690 --> 00:33:32.100 So what do we end up with? 00:33:42.670 --> 00:33:44.440 Let's imagine our chain looks like this. 00:33:51.750 --> 00:33:53.700 What do we see? 00:33:53.700 --> 00:33:57.330 So two carbons are added during each elongation cycle 00:33:57.330 --> 00:34:03.570 to the chain here, and we can see those here, here, 00:34:03.570 --> 00:34:07.060 here, and here. 00:34:07.060 --> 00:34:08.280 OK? 00:34:08.280 --> 00:34:11.219 So a total of four C2 units, one from the starter 00:34:11.219 --> 00:34:14.070 and then three from these three extenders. 00:34:14.070 --> 00:34:17.310 And then we can look at what the functional group status is 00:34:17.310 --> 00:34:23.595 and say, OK, well here, we have no ketoreductase. 00:34:26.719 --> 00:34:30.560 And here, there was ketoreductase action, 00:34:30.560 --> 00:34:31.880 but there's no dehydratase. 00:34:37.090 --> 00:34:39.920 And here, what do we see? 00:34:39.920 --> 00:34:42.940 We see that there was a reduction of the beta-ketone 00:34:42.940 --> 00:34:45.040 and then the action of the dehydratase, 00:34:45.040 --> 00:34:52.060 but we're left at the alkene, so no enoyl reductase. 00:34:52.060 --> 00:34:52.560 Right? 00:34:52.560 --> 00:34:55.219 So just looking, you can begin to decipher 00:34:55.219 --> 00:34:58.260 in a given module what optional domains are there. 00:35:01.030 --> 00:35:06.270 So what we'll do is take a look at an actual PKS assembly line 00:35:06.270 --> 00:35:09.840 and then look at the chemistry happening on it here. 00:35:09.840 --> 00:35:11.565 These are just for your review. 00:35:14.260 --> 00:35:19.560 This is a polyketide synthase responsible for making 00:35:19.560 --> 00:35:24.010 this molecule here. 00:35:24.010 --> 00:35:30.210 So D-E-B or DEB is a 14-membered macrolactone. 00:35:30.210 --> 00:35:35.910 It's a precursor to the antibiotic erythromycin here, 00:35:35.910 --> 00:35:38.340 and this is the cartoon depiction 00:35:38.340 --> 00:35:42.900 of the polyketide synthase required for the biosynthesis 00:35:42.900 --> 00:35:45.960 of this molecule. 00:35:45.960 --> 00:35:52.020 So what do we see looking at this polyketide synthase? 00:35:52.020 --> 00:35:55.230 So it's more complicated than this one here, 00:35:55.230 --> 00:35:58.110 but the same principles apply. 00:35:58.110 --> 00:36:01.980 And what we'll see is that it's comprised of three proteins. 00:36:01.980 --> 00:36:06.210 There's seven modules, so one loading or starter module 00:36:06.210 --> 00:36:12.900 and six elongation modules, and there's a total of 28 domains. 00:36:12.900 --> 00:36:13.620 OK? 00:36:13.620 --> 00:36:16.920 And I said before, the placement and the identity 00:36:16.920 --> 00:36:20.340 of these domains dictates the identity of the growing chain. 00:36:20.340 --> 00:36:22.080 So let's take a look. 00:36:22.080 --> 00:36:26.100 So first, how do we know there's three proteins? 00:36:26.100 --> 00:36:28.410 We know that in this type of cartoon 00:36:28.410 --> 00:36:31.170 because we end up seeing some breaks 00:36:31.170 --> 00:36:33.370 between different domains. 00:36:33.370 --> 00:36:37.830 So here, for instance, the AT, the T, the KS, et cetera, 00:36:37.830 --> 00:36:40.680 they're all attached to one another in the cartoon. 00:36:40.680 --> 00:36:42.870 That means it's all one polypeptide chain, 00:36:42.870 --> 00:36:44.730 but this one polypeptide chain has 00:36:44.730 --> 00:36:47.490 many different enzymatic activities in it, 00:36:47.490 --> 00:36:49.740 because it has different domains. 00:36:49.740 --> 00:36:50.790 When we see a break-- 00:36:50.790 --> 00:36:54.540 so for instance here this T domain and this KS domain 00:36:54.540 --> 00:36:56.250 are not touching one another. 00:36:56.250 --> 00:36:59.410 That means we have two separate proteins. 00:36:59.410 --> 00:37:03.540 So this T domain is at the terminus of DEBS 1, 00:37:03.540 --> 00:37:06.880 and DEBS 2 begins with this ketosynthase. 00:37:06.880 --> 00:37:07.650 OK? 00:37:07.650 --> 00:37:11.070 Likewise, we have a break here, between the T domain 00:37:11.070 --> 00:37:12.930 and this ketosynthase. 00:37:12.930 --> 00:37:16.642 So three proteins make up this assembly line, 00:37:16.642 --> 00:37:18.600 and so when thinking about this, these proteins 00:37:18.600 --> 00:37:21.360 are going to have to interact with each other in one 00:37:21.360 --> 00:37:22.450 way or another. 00:37:22.450 --> 00:37:25.560 And so there's a lot of dynamics in protein-protein interactions 00:37:25.560 --> 00:37:27.570 happening here. 00:37:27.570 --> 00:37:30.090 How do we know there's seven modules? 00:37:30.090 --> 00:37:36.300 And remember each module is responsible for one monomer 00:37:36.300 --> 00:37:37.110 unit. 00:37:37.110 --> 00:37:41.558 We count the T domains, so we have one, two, three, four, 00:37:41.558 --> 00:37:44.910 five, six, seven T domains. 00:37:44.910 --> 00:37:46.700 So like the acyl carrier proteins 00:37:46.700 --> 00:37:49.680 of fatty acid synthase, these T domains 00:37:49.680 --> 00:37:53.400 will be post-translationally modified with a PPant arm. 00:37:53.400 --> 00:37:55.890 And that PPant arm will be loaded 00:37:55.890 --> 00:38:00.690 with the acetyl-CoA or methylmalonyl-CoA or 00:38:00.690 --> 00:38:03.960 malonyl-CoA monomers. 00:38:03.960 --> 00:38:05.500 We have a loading module. 00:38:05.500 --> 00:38:07.890 So the loading module has no ketosynthase, 00:38:07.890 --> 00:38:10.860 because there's nothing upstream over here 00:38:10.860 --> 00:38:14.000 for catalyzing a carbon-carbon bond formation event. 00:38:14.000 --> 00:38:16.710 And then we see modules one through six, 00:38:16.710 --> 00:38:21.090 so sometimes the loading module is module zero. 00:38:21.090 --> 00:38:25.050 We see that each one has a ketosynthase, 00:38:25.050 --> 00:38:27.360 so there'll be carbon-carbon bond formation 00:38:27.360 --> 00:38:29.550 going along this assembly line. 00:38:29.550 --> 00:38:32.700 And we see that the optional domains vary. 00:38:32.700 --> 00:38:36.390 So for instance, module one has a ketoreductase 00:38:36.390 --> 00:38:38.580 as does module two. 00:38:38.580 --> 00:38:39.720 Look at module four. 00:38:39.720 --> 00:38:43.140 We see all three domains required 00:38:43.140 --> 00:38:48.270 for complete processing of that beta-keto group here. 00:38:48.270 --> 00:38:52.570 Here, only a ketoreductase, and here only a ketoreductase. 00:38:52.570 --> 00:38:53.070 OK? 00:38:53.070 --> 00:38:54.880 So just looking at this, you can say, 00:38:54.880 --> 00:38:58.680 OK well, we'll have an OH group here, here. 00:38:58.680 --> 00:39:00.870 Here we have complete processing. 00:39:00.870 --> 00:39:01.830 Just ignore this. 00:39:01.830 --> 00:39:06.310 It's in lower case, because it's a non-functional reductase 00:39:06.310 --> 00:39:07.200 domain. 00:39:07.200 --> 00:39:12.000 It's not operating as annotated here. 00:39:12.000 --> 00:39:15.310 So what happens? 00:39:15.310 --> 00:39:18.300 So again, there's post-translational modification 00:39:18.300 --> 00:39:21.530 of this T domain, so it has a serine. 00:39:21.530 --> 00:39:26.160 The serine gets modified with the PPant arm, as shown here, 00:39:26.160 --> 00:39:28.920 and we use that squiggle depiction, 00:39:28.920 --> 00:39:33.630 as I showed for the acyl carrier protein of FAS. 00:39:33.630 --> 00:39:36.510 So post-translational modification of these T domains 00:39:36.510 --> 00:39:39.390 has to happen before any of the monomers 00:39:39.390 --> 00:39:41.910 are loaded onto this assembly line. 00:39:41.910 --> 00:39:47.580 And these PPant arms allow us to use bioesters as the linkages 00:39:47.580 --> 00:39:50.470 and through the chemistry I showed earlier. 00:39:50.470 --> 00:39:56.100 So here, what we're seeing in this cartoon, going from here, 00:39:56.100 --> 00:39:58.560 this indicates that the T domains are not 00:39:58.560 --> 00:40:00.940 post-translationally modified. 00:40:00.940 --> 00:40:04.080 And here, we see the assembly line 00:40:04.080 --> 00:40:08.460 after action of some [? phosphopentyltransferase ?] 00:40:08.460 --> 00:40:10.850 loading these arms. 00:40:10.850 --> 00:40:11.430 OK? 00:40:11.430 --> 00:40:16.380 So each T domain gets post-translationally modified. 00:40:16.380 --> 00:40:17.910 What happens next? 00:40:17.910 --> 00:40:20.220 We have loading of monomers. 00:40:20.220 --> 00:40:23.040 And we'll look at module zero and one on the board 00:40:23.040 --> 00:40:26.220 and then look at how the whole assembly line goes. 00:40:30.945 --> 00:40:32.590 AUDIENCE: Do you ever get selected 00:40:32.590 --> 00:40:36.260 post-translational modification of the T domains 00:40:36.260 --> 00:40:39.290 and if so, does that facilitate different modules being 00:40:39.290 --> 00:40:40.950 like on or off, so to speak? 00:40:40.950 --> 00:40:42.200 ELIZABETH NOLAN: I don't know. 00:40:42.200 --> 00:40:45.050 I don't know in terms of the kinetics, 00:40:45.050 --> 00:40:48.860 and say, does one T domain get loaded by a PPTase 00:40:48.860 --> 00:40:52.430 before the other? 00:40:52.430 --> 00:40:56.090 These enzymes are very complex, and there's 00:40:56.090 --> 00:40:58.040 a lot we don't know. 00:40:58.040 --> 00:41:01.370 But that would be interesting, if it's the case. 00:41:01.370 --> 00:41:04.784 I wouldn't rule it out, but I just don't know. 00:41:04.784 --> 00:41:10.700 One thing to point out too, these assembly lines are huge. 00:41:10.700 --> 00:41:14.690 So this is something we'll talk about more the next time, 00:41:14.690 --> 00:41:18.690 as we begin to discuss how do you experimentally study them? 00:41:18.690 --> 00:41:23.030 But some are the size of the ribosome for the biosynthesis 00:41:23.030 --> 00:41:25.200 of one natural product. 00:41:25.200 --> 00:41:27.050 And what that means, from the standpoint 00:41:27.050 --> 00:41:29.420 of in vitro characterization, is that often 00:41:29.420 --> 00:41:33.350 you just can't express a whole assembly line, 00:41:33.350 --> 00:41:36.800 let alone say one protein that has a few modules. 00:41:36.800 --> 00:41:40.790 So often, what people will do is individually express domains 00:41:40.790 --> 00:41:43.910 or dye domains and study the reactions 00:41:43.910 --> 00:41:46.560 they catalyze in their chemistry there. 00:41:46.560 --> 00:41:49.640 And so it would be very difficult even 00:41:49.640 --> 00:41:52.850 to test that in terms of in vitro. 00:41:52.850 --> 00:41:56.470 Is there an ordering to how the T domains are loaded? 00:41:56.470 --> 00:41:58.220 And then there's question too, do you even 00:41:58.220 --> 00:42:01.310 know what the dedicated PPTase is? 00:42:01.310 --> 00:42:04.310 So there's some tricks that are done on the bench top 00:42:04.310 --> 00:42:07.640 to get around not knowing that, which we'll talk about later. 00:42:07.640 --> 00:42:22.210 So back to this assembly line to make DEB. 00:42:22.210 --> 00:42:25.180 So we're just going to go over the loading module 00:42:25.180 --> 00:42:30.160 and module 1 and look at a Claisen condensation catalyzed 00:42:30.160 --> 00:42:31.420 by the KS. 00:42:31.420 --> 00:42:36.310 And this chemistry pertains to the various other modules 00:42:36.310 --> 00:42:37.940 and other PKS. 00:42:37.940 --> 00:42:48.600 So we have our AT domain and our thiolation domain of module 0, 00:42:48.600 --> 00:42:57.950 and then we have the ketosynthase, the AT domain, 00:42:57.950 --> 00:43:06.950 the ketoreductase, and the T domain of module 1. 00:43:06.950 --> 00:43:07.450 OK. 00:43:07.450 --> 00:43:09.610 I'm drawing these a little up and down just 00:43:09.610 --> 00:43:11.710 to make it easier to show the chemistry. 00:43:11.710 --> 00:43:13.180 So sometimes you see them straight, 00:43:13.180 --> 00:43:16.870 sometimes moved around here, but it's all the same. 00:43:16.870 --> 00:43:24.760 So we have these PPant arms on the two T domains. 00:43:24.760 --> 00:43:28.270 So what happens now, after these have been post-translationally 00:43:28.270 --> 00:43:29.650 modified? 00:43:29.650 --> 00:43:32.140 We need the action of the AT domains 00:43:32.140 --> 00:43:36.160 to load the monomers onto the PPant arms 00:43:36.160 --> 00:43:42.800 here, so action of the AT domain. 00:44:10.780 --> 00:44:12.040 So what do we end up with? 00:44:12.040 --> 00:44:18.900 In this case, the starter is a propionyl-CoA, 00:44:18.900 --> 00:44:20.430 so we can see that here. 00:44:30.530 --> 00:44:36.430 And we have a methylmalonyl-CoA as the extender, that 00:44:36.430 --> 00:44:46.420 gets loaded, and I'm going to draw the cysteine thiolate 00:44:46.420 --> 00:44:49.870 of the ketosynthase here. 00:44:49.870 --> 00:44:51.430 So what happens next? 00:44:54.820 --> 00:44:59.350 We need to have decarboxylation of the methylmalonyl-CoA 00:44:59.350 --> 00:45:02.440 monomer to give us a C3 unit. 00:45:02.440 --> 00:45:05.140 And it's C3 because of this methyl group, 00:45:05.140 --> 00:45:07.840 but the growing chain will grow by two carbons. 00:45:07.840 --> 00:45:12.070 And then we need to have transfer of this starter 00:45:12.070 --> 00:45:13.630 to the ketosynthase. 00:45:13.630 --> 00:45:18.220 So the ketosynthase is involved in covalent catalysis here. 00:45:18.220 --> 00:45:24.190 So what happens, we can imagine here, we have attack, 00:45:24.190 --> 00:45:27.640 and then here, we're going to have the decarboxylation. 00:45:55.730 --> 00:46:03.860 We have chain transfer to the ketosynthase, 00:46:03.860 --> 00:46:12.330 and here, decarboxylation leaves us this species. 00:46:12.330 --> 00:46:12.830 OK? 00:46:51.810 --> 00:46:53.340 OK. 00:46:53.340 --> 00:46:54.630 So now, what happens? 00:46:54.630 --> 00:46:59.940 Now, the assembly's set up for the Claisen condensation 00:46:59.940 --> 00:47:05.530 to occur which is catalyzed by the ketosynthase. 00:47:05.530 --> 00:47:06.030 Right? 00:47:06.030 --> 00:47:08.080 So what will happen here? 00:47:08.080 --> 00:47:17.490 You can imagine that, and as a result, where do we end up? 00:47:17.490 --> 00:47:18.840 I'll just draw it down here. 00:47:49.010 --> 00:47:50.120 And what else do we have? 00:47:50.120 --> 00:47:55.760 We have a ketoreductase. 00:47:55.760 --> 00:47:59.900 So this ketoreductase will act on the monomer 00:47:59.900 --> 00:48:03.110 of the upstream unit, and that's how it always is. 00:48:03.110 --> 00:48:07.130 So if there's optional domains in module 1, 00:48:07.130 --> 00:48:10.490 they act on the monomer from module 0. 00:48:10.490 --> 00:48:13.160 If there's optional domains in module 2, 00:48:13.160 --> 00:48:15.740 they'll act on the monomer for module 1. 00:48:15.740 --> 00:48:16.400 OK? 00:48:16.400 --> 00:48:20.330 So we see here now we have reduction 00:48:20.330 --> 00:48:26.080 of the ketone from module 1 to here via the ketoreductase. 00:48:26.080 --> 00:48:26.580 OK? 00:48:48.960 --> 00:48:58.530 So if we take a look at what's on the PowerPoint 00:48:58.530 --> 00:49:03.210 here, what we're seeing is one depiction of this assembly line 00:49:03.210 --> 00:49:07.720 to make DEB indicating the growing chain. 00:49:07.720 --> 00:49:08.220 OK? 00:49:08.220 --> 00:49:11.550 So as we walk through each module, 00:49:11.550 --> 00:49:14.850 we see an additional monomer attached. 00:49:14.850 --> 00:49:16.650 So the chain elongates, and then you 00:49:16.650 --> 00:49:21.690 can track what's happening to the ketone group 00:49:21.690 --> 00:49:25.080 of the upstream monomer on the basis of the optional domains 00:49:25.080 --> 00:49:27.300 here. 00:49:27.300 --> 00:49:30.930 If we look in this one, which I like this one because they 00:49:30.930 --> 00:49:32.250 color code. 00:49:32.250 --> 00:49:36.730 So they color code the different modules along with the monomer, 00:49:36.730 --> 00:49:39.640 and so it's pretty easy to trace what's happening. 00:49:39.640 --> 00:49:44.080 So for instance, here we have the loading module, 00:49:44.080 --> 00:49:46.980 and we have the starter unit in red. 00:49:46.980 --> 00:49:50.400 And here we see that it's been reduced by the ketoreductase 00:49:50.400 --> 00:49:52.650 of the upstream blue module. 00:49:52.650 --> 00:49:55.590 Here, we have the green module, here is its monomer, 00:49:55.590 --> 00:49:59.670 and we see its ketoreductase acted on the blue monomer 00:49:59.670 --> 00:50:02.040 from module 1, et cetera here. 00:50:02.040 --> 00:50:05.730 So I encourage you all to just very systematically work 00:50:05.730 --> 00:50:10.260 through the assembly lines that are provided in these notes, 00:50:10.260 --> 00:50:13.990 and it's the same type of chemistry over and over again. 00:50:13.990 --> 00:50:15.420 And if you learn the patterns, it 00:50:15.420 --> 00:50:18.540 ends up being quite easy to work through, at least 00:50:18.540 --> 00:50:19.990 the simple assembly line. 00:50:19.990 --> 00:50:23.470 So as you can imagine, complexity increases, 00:50:23.470 --> 00:50:26.760 and we'll look at some examples of more complex ones as well. 00:50:26.760 --> 00:50:29.040 So where we'll start next time with this 00:50:29.040 --> 00:50:33.720 is just briefly looking at chain release by the thioesterase. 00:50:33.720 --> 00:50:37.320 And then we'll do an overview of non-ribosomal peptide 00:50:37.320 --> 00:50:42.370 biosynthesis logic and then look at some example assembly lines. 00:50:42.370 --> 00:50:46.620 So we have the exams to give back. 00:50:46.620 --> 00:50:48.840 I'll just say a few things. 00:50:48.840 --> 00:50:56.090 So the average was around a 68, plus or minus 10, 11, 00:50:56.090 --> 00:50:58.530 12 for the standard deviation. 00:50:58.530 --> 00:51:01.560 I'd say, if you were in the mid 70s and above, 00:51:01.560 --> 00:51:03.390 you did really well. 00:51:03.390 --> 00:51:07.500 If you're into the low 60s, that is OK, 00:51:07.500 --> 00:51:12.180 but we'd really like things to improve for the next one. 00:51:12.180 --> 00:51:17.205 In terms of the exam and just some feedback-- and I'll 00:51:17.205 --> 00:51:20.160 put feedback as well in the key which will be posted 00:51:20.160 --> 00:51:21.990 later today or early tomorrow. 00:51:21.990 --> 00:51:25.880 There wasn't one question that say the whole class bombed, 00:51:25.880 --> 00:51:27.180 so that's good. 00:51:27.180 --> 00:51:29.552 There were a few things for just general improvement, 00:51:29.552 --> 00:51:32.010 and I want to bring this up, so you can also think about it 00:51:32.010 --> 00:51:33.900 in terms of problem sets. 00:51:33.900 --> 00:51:37.420 One involves being quantitative. 00:51:37.420 --> 00:51:41.080 So there's certainly qualitative trends and data, 00:51:41.080 --> 00:51:44.340 but there's also quantitative information there, 00:51:44.340 --> 00:51:46.830 and that can be important to look at. 00:51:46.830 --> 00:51:51.030 And one example I'll give of that involved question one. 00:51:51.030 --> 00:51:55.080 If you recall, there was an analysis of GDP hydrolysis 00:51:55.080 --> 00:51:58.260 and an analysis of peptide bond formation. 00:51:58.260 --> 00:52:03.360 And quantitative analysis of the peptide bond formation 00:52:03.360 --> 00:52:08.850 experiments will show that all of the lysyl-tRNAs 00:52:08.850 --> 00:52:14.130 were used up in the case of the codon that was AAA. 00:52:14.130 --> 00:52:16.800 Whereas, some of those tRNAs were not 00:52:16.800 --> 00:52:22.710 used up when the codon contained that 6-methyl-A 00:52:22.710 --> 00:52:24.370 in position one. 00:52:24.370 --> 00:52:24.870 Right? 00:52:24.870 --> 00:52:28.200 And if you linked that back to the kinetic model 00:52:28.200 --> 00:52:30.240 along with the other data, what that indicates 00:52:30.240 --> 00:52:31.840 is that proofreading is going on. 00:52:31.840 --> 00:52:32.340 Right? 00:52:32.340 --> 00:52:37.050 Some of those tRNAs are being rejected from the ribosome 00:52:37.050 --> 00:52:37.740 there. 00:52:37.740 --> 00:52:41.160 So that was one place where quantitiation, a fair number 00:52:41.160 --> 00:52:43.570 of you missed that. 00:52:43.570 --> 00:52:45.300 And another thing I just want to stress 00:52:45.300 --> 00:52:49.740 is to make sure you answered the question being asked. 00:52:49.740 --> 00:52:54.300 And where an example of that came up was in question one 00:52:54.300 --> 00:52:58.140 with the final question asking about relating the data back 00:52:58.140 --> 00:52:59.997 to the kinetic model. 00:52:59.997 --> 00:53:01.830 And so if a question asks that you really do 00:53:01.830 --> 00:53:04.470 need to go back to the model which was in the appendix 00:53:04.470 --> 00:53:05.970 and think about that. 00:53:05.970 --> 00:53:08.490 So many of you gave some very interesting answers 00:53:08.490 --> 00:53:12.495 and presented hypotheses about perhaps the 6-methyl-A 00:53:12.495 --> 00:53:15.225 is involved in regulation and controlling 00:53:15.225 --> 00:53:17.780 like the timing of translation. 00:53:17.780 --> 00:53:20.680 And that's terrific and interesting to think about, 00:53:20.680 --> 00:53:22.660 but it wasn't the answer to the question. 00:53:22.660 --> 00:53:23.160 Right? 00:53:23.160 --> 00:53:26.130 Which was to go beyond the conclusions 00:53:26.130 --> 00:53:29.100 from the experiments with GTP hydrolysis 00:53:29.100 --> 00:53:32.910 and formation of that dipeptide, and ask 00:53:32.910 --> 00:53:35.580 how can we conceptualize this from the standpoint 00:53:35.580 --> 00:53:37.950 of the model we studied in class? 00:53:37.950 --> 00:53:41.280 And then just the third point I'll make 00:53:41.280 --> 00:53:49.080 is related to question two and specifically to GroEL. 00:53:49.080 --> 00:53:52.560 But the more general thing is that if we 00:53:52.560 --> 00:53:55.110 learn about a system in class, unless there's 00:53:55.110 --> 00:53:57.150 compelling data presented in a question 00:53:57.150 --> 00:54:00.263 to suggest the model is something other than what we 00:54:00.263 --> 00:54:02.430 learned or its behavior is something other than what 00:54:02.430 --> 00:54:05.550 we learned, stick with what you know. 00:54:05.550 --> 00:54:10.530 So in the use of GroEL, the idea in that experiment 00:54:10.530 --> 00:54:14.640 was that, if you recall, this question was looking at these J 00:54:14.640 --> 00:54:17.730 proteins and asking, how do J proteins 00:54:17.730 --> 00:54:20.050 facilitate disaggregation? 00:54:20.050 --> 00:54:20.550 Right? 00:54:20.550 --> 00:54:26.070 And so a GroEL trap was used that cannot hydrolyze ATP, 00:54:26.070 --> 00:54:33.240 which means it's not active at folding any polypeptide. 00:54:33.240 --> 00:54:37.170 But the idea there is that these J proteins end up 00:54:37.170 --> 00:54:40.320 allowing monomers to come out of the aggregate, 00:54:40.320 --> 00:54:43.320 and then GroEL can trap and unfold the monomer 00:54:43.320 --> 00:54:46.290 to prevent reactivation. 00:54:46.290 --> 00:54:49.200 And so a number of people came to the conclusion 00:54:49.200 --> 00:54:52.500 that GroEL was binding that aggregate somehow 00:54:52.500 --> 00:54:53.580 in its chamber. 00:54:53.580 --> 00:54:55.200 And what we learned about GroEL is 00:54:55.200 --> 00:54:59.060 that its chamber can't house a protein over 60 kilodaltons. 00:54:59.060 --> 00:54:59.560 Right? 00:54:59.560 --> 00:55:01.800 We saw that in terms of the in vitro assays 00:55:01.800 --> 00:55:05.010 that were done looking at what its native substrates are. 00:55:05.010 --> 00:55:05.510 Right? 00:55:05.510 --> 00:55:07.950 So always go back to what you know, and then you 00:55:07.950 --> 00:55:10.080 need to ask yourselves, are the data 00:55:10.080 --> 00:55:12.950 suggesting some other behavior? 00:55:12.950 --> 00:55:15.050 And if that were the case, like what 00:55:15.050 --> 00:55:18.070 is your analysis of those data there? 00:55:18.070 --> 00:55:22.530 So please, even if you did really well, look at the key 00:55:22.530 --> 00:55:24.107 and see what the key has to say. 00:55:24.107 --> 00:55:26.690 And if you have questions, you can make an appointment with me 00:55:26.690 --> 00:55:30.970 or come to office hours or discuss with Shiva there. 00:55:30.970 --> 00:55:32.520 OK?