1 00:00:01,270 --> 00:00:03,700 The following content is provided under a Creative 2 00:00:03,700 --> 00:00:05,240 Commons license. 3 00:00:05,240 --> 00:00:07,540 Your support will help MIT OpenCourseWare 4 00:00:07,540 --> 00:00:11,900 continue to offer high-quality educational resources for free. 5 00:00:11,900 --> 00:00:14,530 To make a donation or view additional materials 6 00:00:14,530 --> 00:00:18,470 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:18,470 --> 00:00:19,660 at ocw.mit.edu. 8 00:00:26,630 --> 00:00:30,020 ELIZABETH NOLAN: We're going to get started 9 00:00:30,020 --> 00:00:33,200 and what we'll do today is continue 10 00:00:33,200 --> 00:00:35,180 with fatty acid synthase. 11 00:00:35,180 --> 00:00:39,350 Because that's the paradigm for these macromolecular machines, 12 00:00:39,350 --> 00:00:42,410 like the PKS, and then we'll go over 13 00:00:42,410 --> 00:00:44,630 the logic of polyketide synthases. 14 00:00:44,630 --> 00:00:50,120 So we left off last time with this discussion 15 00:00:50,120 --> 00:00:52,940 about some molecules that will be involved 16 00:00:52,940 --> 00:00:55,880 and in particular thioesters, and I 17 00:00:55,880 --> 00:01:01,160 asked about the alpha H. So just going back 18 00:01:01,160 --> 00:01:03,630 to introductory organic chemistry, what 19 00:01:03,630 --> 00:01:09,032 are the properties of this atom here? 20 00:01:09,032 --> 00:01:10,240 AUDIENCE: [INAUDIBLE] acidic. 21 00:01:10,240 --> 00:01:11,157 ELIZABETH NOLAN: Yeah. 22 00:01:11,157 --> 00:01:11,660 OK, right. 23 00:01:11,660 --> 00:01:13,190 So this is acidic. 24 00:01:13,190 --> 00:01:14,255 So if you have-- 25 00:01:24,170 --> 00:01:24,670 OK? 26 00:01:24,670 --> 00:01:28,120 So what that means is if there is a base that can deprotonate 27 00:01:28,120 --> 00:01:30,240 that, we can get an enolate. 28 00:01:30,240 --> 00:01:31,840 OK, and this is the type of chemistry 29 00:01:31,840 --> 00:01:35,170 that's going to be happening with the thioesters that 30 00:01:35,170 --> 00:01:40,060 are used in fatty acid synthase and also polyketide synthase. 31 00:01:40,060 --> 00:01:42,490 And just to rewind a little bit more, 32 00:01:42,490 --> 00:01:45,730 if we think about carbon-carbon bond forming reactions 33 00:01:45,730 --> 00:01:48,790 in nature, which is what's happening in fatty acid 34 00:01:48,790 --> 00:01:51,580 biosynthesis and in polyketide biosynthesis, 35 00:01:51,580 --> 00:01:55,690 effectively, nature uses three different types of reaction. 36 00:01:55,690 --> 00:01:58,720 OK, so one is the aldol, two are the Claisen, 37 00:01:58,720 --> 00:02:00,230 and three [INAUDIBLE] transfer. 38 00:02:00,230 --> 00:02:04,420 OK, and so we're going to see Claisen condensations in FAS 39 00:02:04,420 --> 00:02:06,820 and PKS biosynthesis. 40 00:02:06,820 --> 00:02:09,130 And then after spring break, when 41 00:02:09,130 --> 00:02:14,123 Joanne starts with cholesterol biosynthesis, 42 00:02:14,123 --> 00:02:15,790 that will involve [INAUDIBLE] transfers. 43 00:02:15,790 --> 00:02:19,030 And hopefully, you've seen aldol reactions sometime 44 00:02:19,030 --> 00:02:21,640 before within biochemistry here. 45 00:02:21,640 --> 00:02:22,330 OK? 46 00:02:22,330 --> 00:02:25,630 So we need to think about just what the general Claisen 47 00:02:25,630 --> 00:02:27,910 condensation is that we're going to be seeing here 48 00:02:27,910 --> 00:02:30,010 and the consequences of this acidic proton. 49 00:02:33,160 --> 00:02:36,160 So also just keep in mind, rewinding a little more, 50 00:02:36,160 --> 00:02:40,350 nature uses thioesters not esters, 51 00:02:40,350 --> 00:02:43,650 and so the alpha H is more acidic. 52 00:02:43,650 --> 00:02:46,780 The carbonyl is more activated for nucloephilic attack. 53 00:02:46,780 --> 00:02:48,720 And there's some resonance arguments 54 00:02:48,720 --> 00:02:51,880 and orbital overlap arguments that 55 00:02:51,880 --> 00:02:56,320 can guide those conclusions, if you wish to do them here. 56 00:02:56,320 --> 00:02:56,820 OK. 57 00:02:56,820 --> 00:03:03,983 So let's imagine that we have a thioester. 58 00:03:09,540 --> 00:03:10,515 We have a base. 59 00:03:15,510 --> 00:03:22,820 OK, that's going to be [INAUDIBLE],, which 60 00:03:22,820 --> 00:03:24,240 is going to get us to here. 61 00:03:33,840 --> 00:03:41,886 So this is our nucleophile, and what you'll see coming forward 62 00:03:41,886 --> 00:03:43,870 is an enolate. 63 00:03:43,870 --> 00:03:50,188 So imagine we have that, and we add it with another thioester, 64 00:03:50,188 --> 00:03:51,990 and here's our electrophile. 65 00:03:56,760 --> 00:03:58,320 What do we get? 66 00:04:09,320 --> 00:04:23,250 We get formation of a beta-keto thioester, which is the Claisen 67 00:04:23,250 --> 00:04:24,440 condensation product. 68 00:04:34,903 --> 00:04:37,383 OK, you have two thioesters. 69 00:04:40,221 --> 00:04:41,170 OK? 70 00:04:41,170 --> 00:04:45,220 So effectively, this acyl thioester is doubly activated, 71 00:04:45,220 --> 00:04:46,630 so it can be-- 72 00:04:46,630 --> 00:04:47,790 did I lose it? 73 00:04:47,790 --> 00:04:51,360 Oh no, problems. 74 00:04:51,360 --> 00:04:52,990 Sorry about that. 75 00:04:52,990 --> 00:04:57,810 It can be activated as an electrophile at the C1 76 00:04:57,810 --> 00:05:00,470 position, so next door to the sulfur. 77 00:05:00,470 --> 00:05:04,090 And it can be activated as a nucleophile at the C2 position 78 00:05:04,090 --> 00:05:04,750 here. 79 00:05:04,750 --> 00:05:06,730 So this is the general chemistry that's 80 00:05:06,730 --> 00:05:09,970 going to be happening by FAS and PKS 81 00:05:09,970 --> 00:05:12,640 in terms of forming carbon-carbon bonds 82 00:05:12,640 --> 00:05:15,650 between monomers here. 83 00:05:15,650 --> 00:05:16,150 OK? 84 00:05:16,150 --> 00:05:21,180 So in fatty acid synthase, we have two monomer units. 85 00:05:31,730 --> 00:05:32,680 OK? 86 00:05:32,680 --> 00:05:36,400 So we have acetyl-CoA and malonyl-CoA. 87 00:05:49,530 --> 00:06:01,700 Acetyl-CoA is the starter unit, sometimes called unit 0, 88 00:06:01,700 --> 00:06:05,465 and then malonyl-CoA is the extender. 89 00:06:28,340 --> 00:06:32,220 And so recall that in fatty acid biosynthesis, 90 00:06:32,220 --> 00:06:36,690 each elongation event adds two carbons, 91 00:06:36,690 --> 00:06:39,640 and if we look at malonyl-CoA, we have three here. 92 00:06:39,640 --> 00:06:40,140 Right? 93 00:06:40,140 --> 00:06:43,470 So there's decarboxylation of malonyl-CoA 94 00:06:43,470 --> 00:06:55,870 to generate a C2 unit, and there's 95 00:06:55,870 --> 00:06:58,010 details of that in the lecture 15 notes. 96 00:07:07,450 --> 00:07:19,990 And SCoA is coenzyme A, here, and there's some information 97 00:07:19,990 --> 00:07:23,140 as to the biosynthesis of these starter and extender units 98 00:07:23,140 --> 00:07:23,740 in the notes. 99 00:07:23,740 --> 00:07:26,840 We're not going to go over that in lecture here. 100 00:07:26,840 --> 00:07:32,710 So in terms of using these monomers to obtain 101 00:07:32,710 --> 00:07:35,620 fatty acids, first what we're going to go over 102 00:07:35,620 --> 00:07:39,390 are the domains in FAS. 103 00:07:39,390 --> 00:07:41,590 And so we can consider domains that 104 00:07:41,590 --> 00:07:45,880 are required for extension of the fatty acid chain 105 00:07:45,880 --> 00:07:47,590 and then domains that are required 106 00:07:47,590 --> 00:07:52,680 for tailoring of that effectively to reduce 107 00:07:52,680 --> 00:07:57,100 the carbonyl, as shown. 108 00:07:57,100 --> 00:07:58,600 And we're going to go through these, 109 00:07:58,600 --> 00:08:00,058 because what we're going to find is 110 00:08:00,058 --> 00:08:03,670 that with polyketide biosynthesis, 111 00:08:03,670 --> 00:08:06,170 the same types of domains are used. 112 00:08:06,170 --> 00:08:08,790 So this logic extends there. 113 00:08:08,790 --> 00:08:09,290 OK. 114 00:08:09,290 --> 00:08:18,730 So first, we have domains required 115 00:08:18,730 --> 00:08:36,539 for elongation of the fatty acid chain by one two-carbon unit. 116 00:08:39,669 --> 00:08:40,169 OK. 117 00:08:40,169 --> 00:08:44,430 So these include domains that may be abbreviated 118 00:08:44,430 --> 00:08:58,430 as AAT or MAT, and they can be grouped as AT 119 00:08:58,430 --> 00:09:05,955 and stand for acetyl or malonyltransferase. 120 00:09:15,000 --> 00:09:15,540 OK. 121 00:09:15,540 --> 00:09:30,370 We have an Acyl Carrier Protein, ACP, 122 00:09:30,370 --> 00:09:41,390 and this carries the growing chain between the domains 123 00:09:41,390 --> 00:09:43,590 of fatty acid synthase. 124 00:09:43,590 --> 00:09:45,690 And so in recitation this week, you're 125 00:09:45,690 --> 00:09:49,530 going to see how these domains move around 126 00:09:49,530 --> 00:09:54,000 and talk about the length of this acyl carrier protein. 127 00:09:54,000 --> 00:09:55,830 We also have the ketosynthase. 128 00:10:03,450 --> 00:10:06,540 So what the ketosynthase does is it accepts the growing 129 00:10:06,540 --> 00:10:18,460 chain from the acyl carrier protein, 130 00:10:18,460 --> 00:10:31,818 and it catalyzes the Claisen condensation 131 00:10:31,818 --> 00:10:32,735 with the next monomer. 132 00:10:41,660 --> 00:10:43,790 And what we'll see is that this ketosynthase 133 00:10:43,790 --> 00:11:01,000 uses covalent catalysis, and via a cysteine thiolate residue. 134 00:11:04,440 --> 00:11:07,430 So these are the key domains required 135 00:11:07,430 --> 00:11:09,560 for elongation of the chain. 136 00:11:09,560 --> 00:11:10,060 OK? 137 00:11:10,060 --> 00:11:17,240 And then what we also need are domains required for tailoring, 138 00:11:17,240 --> 00:11:22,460 and just to clarify, I'm defining domain here 139 00:11:22,460 --> 00:11:27,800 as a polypeptide with a single enzymatic activity. 140 00:11:27,800 --> 00:11:30,650 So domains can be connected to one another, 141 00:11:30,650 --> 00:11:35,030 or they can be standalone in different types of synthases, 142 00:11:35,030 --> 00:11:37,250 but domain means polypeptide with 143 00:11:37,250 --> 00:11:38,930 a single enzymatic activity. 144 00:11:46,880 --> 00:11:49,450 So what are the domains required for tailoring? 145 00:11:54,250 --> 00:12:03,520 And these work after addition of the C2 unit 146 00:12:03,520 --> 00:12:05,630 to the growing chain. 147 00:12:05,630 --> 00:12:07,850 So first, there's a ketoreductase. 148 00:12:18,370 --> 00:12:20,960 And as indicated, what this enzyme does 149 00:12:20,960 --> 00:12:40,090 is it reduces the carbonyl of the previous unit to an OH 150 00:12:40,090 --> 00:12:48,100 and uses an NADPH H plus. 151 00:12:48,100 --> 00:12:54,250 We also have the dehydratase here, 152 00:12:54,250 --> 00:13:00,730 and this forms an alpha, beta-alkene from the product 153 00:13:00,730 --> 00:13:03,730 of the ketoreductase action. 154 00:13:03,730 --> 00:13:07,240 And then we have an enoyl reductase 155 00:13:07,240 --> 00:13:18,030 that reduces this alpha, beta-alkene, 156 00:13:18,030 --> 00:13:24,670 and this also requires NADPH H plus here. 157 00:13:24,670 --> 00:13:28,150 And then some fatty synthases use 158 00:13:28,150 --> 00:13:32,020 a domain called a thioesterase for chain release, 159 00:13:32,020 --> 00:13:35,210 and that's noted as TE. 160 00:13:43,930 --> 00:13:49,820 And we'll see thioesterases in the PKS and in our PS sections 161 00:13:49,820 --> 00:13:50,320 here. 162 00:13:52,840 --> 00:13:57,130 So one comment regarding the acyl carrier protein, 163 00:13:57,130 --> 00:14:00,490 and then we'll just look at the fatty acid synthase cycle 164 00:14:00,490 --> 00:14:03,190 and see how these domains are acting. 165 00:14:22,090 --> 00:14:27,010 So in order for the acyl carrier protein 166 00:14:27,010 --> 00:14:29,530 to carry this growing chain, it first 167 00:14:29,530 --> 00:14:32,470 needs to be post-translationally modified 168 00:14:32,470 --> 00:14:34,600 with what's called a PPant arm. 169 00:14:34,600 --> 00:14:38,200 And that arm provides the ability 170 00:14:38,200 --> 00:14:43,240 to have these monomers, or growing chains, linked 171 00:14:43,240 --> 00:14:46,660 via a thioester. 172 00:14:46,660 --> 00:14:50,080 And so just to go over this post-translational 173 00:14:50,080 --> 00:14:56,550 modification, so post-translational modification 174 00:14:56,550 --> 00:15:01,690 of acyl carrier protein with the PPant arm. 175 00:15:05,150 --> 00:15:05,650 OK. 176 00:15:05,650 --> 00:15:13,520 If we consider apo acyl carrier protein, 177 00:15:13,520 --> 00:15:16,420 and apo means that the PPant arm is not attached. 178 00:15:21,720 --> 00:15:22,890 There's a serine residue. 179 00:15:27,030 --> 00:15:35,610 An enzyme called the PPTase comes along, 180 00:15:35,610 --> 00:15:38,070 and it allows for post-translational modification 181 00:15:38,070 --> 00:15:49,410 of this serine using CoASH, releasing 3', 5'-ADP to give 182 00:15:49,410 --> 00:15:55,970 ACP post-translationally modified with the PPant arm. 183 00:15:55,970 --> 00:15:56,470 OK? 184 00:15:56,470 --> 00:15:58,012 And we'll look at the actual chemical 185 00:15:58,012 --> 00:16:00,270 structures in a minute. 186 00:16:00,270 --> 00:16:03,780 What I want to point out is that throughout this unit, 187 00:16:03,780 --> 00:16:07,920 this squiggle, some form of squiggle here, 188 00:16:07,920 --> 00:16:10,320 is the abbreviation for the PPant arm. 189 00:16:18,760 --> 00:16:19,770 OK? 190 00:16:19,770 --> 00:16:28,230 And this is very flexible and about 20 angstroms in length. 191 00:16:35,130 --> 00:16:38,850 So what does this actually look like? 192 00:16:38,850 --> 00:16:41,420 So here we have CoASH. 193 00:16:44,090 --> 00:16:48,840 So PPant is an abbreviation for phosphopantetheine, here, 194 00:16:48,840 --> 00:16:53,220 this moiety, and here's the 3', 5'-ADP. 195 00:16:53,220 --> 00:16:58,410 And so effectively, what's shown on the board is repeated here. 196 00:16:58,410 --> 00:17:01,050 Except for here, we're seeing the full structure 197 00:17:01,050 --> 00:17:04,650 of the phosphopantetheinylated acyl carrier protein. 198 00:17:04,650 --> 00:17:07,560 So this squiggle abbreviation indicates 199 00:17:07,560 --> 00:17:10,050 this post-translational modification 200 00:17:10,050 --> 00:17:11,999 onto a serine residue of the ACP. 201 00:17:14,760 --> 00:17:17,060 Just as an example of structure, so here 202 00:17:17,060 --> 00:17:21,000 is a structure of acyl carrier protein from E. coli. 203 00:17:21,000 --> 00:17:24,060 It's about 10 kilodaltons, so not very big, 204 00:17:24,060 --> 00:17:28,010 and we see the PPant arm here attached. 205 00:17:28,010 --> 00:17:30,330 OK? 206 00:17:30,330 --> 00:17:40,670 So if we think about fatty acids biosynthesis, 207 00:17:40,670 --> 00:17:46,610 we can think about this in three steps, better iterated. 208 00:17:49,410 --> 00:17:49,910 OK. 209 00:17:49,910 --> 00:17:56,810 So first we have loading, so the acyl carrier proteins need 210 00:17:56,810 --> 00:17:59,330 to be loaded with monomers. 211 00:17:59,330 --> 00:18:03,560 Sometimes, this step the reactions 212 00:18:03,560 --> 00:18:05,810 are described as priming reactions. 213 00:18:12,440 --> 00:18:18,690 We have initiation and elongation 214 00:18:18,690 --> 00:18:24,930 all grouped together here and, three, at some point, 215 00:18:24,930 --> 00:18:25,860 a termination. 216 00:18:28,920 --> 00:18:29,740 OK? 217 00:18:29,740 --> 00:18:32,440 So we've thought about these before from the standpoint 218 00:18:32,440 --> 00:18:35,500 of biological polymerizations. 219 00:18:42,650 --> 00:18:46,640 So what about the FAS cycle? 220 00:18:46,640 --> 00:18:51,350 Here's one depiction, and I've provided multiple depictions 221 00:18:51,350 --> 00:18:52,730 in the lecture 15 notes. 222 00:18:52,730 --> 00:18:55,940 Because some people find different cycles easier 223 00:18:55,940 --> 00:18:58,610 than others, but let's just take a look. 224 00:18:58,610 --> 00:19:03,110 So this charts out the various domains-- 225 00:19:03,110 --> 00:19:06,500 the starter and the extender and then the chemistry that 226 00:19:06,500 --> 00:19:08,930 occurs on these steps. 227 00:19:08,930 --> 00:19:12,770 And so what needs to happen is that there 228 00:19:12,770 --> 00:19:16,820 needs to be some loading and initiation where 229 00:19:16,820 --> 00:19:22,370 the acetyl-CoA is loaded onto an acyl carrier protein. 230 00:19:22,370 --> 00:19:27,530 So that's shown here via transferase here, 231 00:19:27,530 --> 00:19:30,230 and then, from the acyl carrier protein, 232 00:19:30,230 --> 00:19:34,340 this monomer is loaded onto the ketosynthase. 233 00:19:34,340 --> 00:19:36,950 If we look here, we have one of our extender units, 234 00:19:36,950 --> 00:19:40,250 the malonyl-CoA, and the CO2 unit 235 00:19:40,250 --> 00:19:42,610 that gets removed during decarboxylation, 236 00:19:42,610 --> 00:19:44,430 as shown in this light blue. 237 00:19:44,430 --> 00:19:44,930 OK? 238 00:19:44,930 --> 00:19:49,580 We need to have this extender unit also transferred 239 00:19:49,580 --> 00:19:53,090 to an acyl carrier protein via the action of an AT. 240 00:19:53,090 --> 00:19:54,800 So we see lots of the CoA. 241 00:19:54,800 --> 00:19:58,040 Here we have the acyl carrier protein with the PPant arm. 242 00:19:58,040 --> 00:19:59,210 It's not a squiggle here. 243 00:19:59,210 --> 00:20:03,110 It is the next one with this malonyl unit loaded. 244 00:20:03,110 --> 00:20:07,490 There's a decarboxylation, and what do we see happening here? 245 00:20:07,490 --> 00:20:10,370 We have a chain elongation event, 246 00:20:10,370 --> 00:20:12,950 so Claisen condensation catalyzed 247 00:20:12,950 --> 00:20:16,760 by the ketosynthase between the starter and the first extender 248 00:20:16,760 --> 00:20:19,280 to give us this beta-keto thioester. 249 00:20:19,280 --> 00:20:21,800 So once this carbon-carbon bond is formed to give us 250 00:20:21,800 --> 00:20:26,000 the beta-keto thioester, there's processing of the beta carbon 251 00:20:26,000 --> 00:20:27,530 via those tailoring domains-- 252 00:20:30,240 --> 00:20:33,050 the dehydratase and the enoyl reductase. 253 00:20:33,050 --> 00:20:38,210 And so we see reduction of the beta ketone here, 254 00:20:38,210 --> 00:20:41,630 we see formation of the alkene, and then we see reduction 255 00:20:41,630 --> 00:20:43,220 to get us to this point. 256 00:20:43,220 --> 00:20:47,720 And so this cycle can repeat itself until, at some point, 257 00:20:47,720 --> 00:20:49,040 there's a termination event. 258 00:20:49,040 --> 00:20:52,850 And in this case here, we see a thioesterase catalyzing 259 00:20:52,850 --> 00:20:57,260 hydrolytic release of the fatty acid chain. 260 00:20:57,260 --> 00:20:59,930 This is the depiction you'll see in recitation today, 261 00:20:59,930 --> 00:21:01,340 or saw before. 262 00:21:01,340 --> 00:21:03,710 And I guess what I like about this depiction is 263 00:21:03,710 --> 00:21:07,070 that you see color coding separating 264 00:21:07,070 --> 00:21:09,740 the elongation and the domains involved 265 00:21:09,740 --> 00:21:14,330 in elongation with then the processing of the beta ketone 266 00:21:14,330 --> 00:21:17,210 here and then termination. 267 00:21:17,210 --> 00:21:18,220 OK. 268 00:21:18,220 --> 00:21:23,170 So we get some fatty acid from this. 269 00:21:23,170 --> 00:21:27,610 And so where we're going to go with this overview is looking 270 00:21:27,610 --> 00:21:32,770 at the polyketides and to ask what similar and different 271 00:21:32,770 --> 00:21:36,700 in terms of polyketide biosynthesis? 272 00:21:36,700 --> 00:21:39,400 And so where we can begin with thinking 273 00:21:39,400 --> 00:21:44,770 about that is asking what are the starters and extenders? 274 00:21:44,770 --> 00:21:47,620 And so these are the starters and extenders 275 00:21:47,620 --> 00:21:50,020 we saw for fatty acids, and here are 276 00:21:50,020 --> 00:21:54,290 the starters and extenders for polyketides, so very similar. 277 00:21:54,290 --> 00:21:54,790 Right? 278 00:21:54,790 --> 00:21:57,010 We just see that there's some additional options, 279 00:21:57,010 --> 00:21:59,630 so we also have this propionyl-CoA here. 280 00:21:59,630 --> 00:22:02,500 In addition to malonyl-CoA as an extender, 281 00:22:02,500 --> 00:22:07,880 we see that methylmalonyl-CoA can be employed. 282 00:22:07,880 --> 00:22:12,170 So what are the core domains of the PKS? 283 00:22:12,170 --> 00:22:16,640 They're similar to those of FAS, and we'll just focus 284 00:22:16,640 --> 00:22:19,020 on the PKS side of this table. 285 00:22:19,020 --> 00:22:21,950 So this is a helpful table when reviewing 286 00:22:21,950 --> 00:22:24,230 both types of assembly lines. 287 00:22:24,230 --> 00:22:27,320 So the core means that every module, 288 00:22:27,320 --> 00:22:31,310 which I'll define in a moment, contains these domains. 289 00:22:31,310 --> 00:22:33,860 So we see that there's a ketosynthase, 290 00:22:33,860 --> 00:22:37,280 an acyltransferase, and a thiolation domain. 291 00:22:37,280 --> 00:22:39,770 So this thiolation domain is the same 292 00:22:39,770 --> 00:22:42,020 as the acyl carrier protein. 293 00:22:42,020 --> 00:22:45,590 So there's different terminology used, and within the notes, 294 00:22:45,590 --> 00:22:49,220 I have some pages that are dedicated 295 00:22:49,220 --> 00:22:52,440 to these terminologies. 296 00:22:52,440 --> 00:22:54,250 OK? 297 00:22:54,250 --> 00:23:03,680 So for PKS, here, we have the ketosynthase, 298 00:23:03,680 --> 00:23:07,610 we have acetyltransferase, and then 299 00:23:07,610 --> 00:23:12,740 we have this T domain which equals acyl carrier 300 00:23:12,740 --> 00:23:14,340 protein here. 301 00:23:14,340 --> 00:23:15,530 OK? 302 00:23:15,530 --> 00:23:17,480 So then what about these tailoring 303 00:23:17,480 --> 00:23:20,930 domains that were required to produce the fatty acid? 304 00:23:20,930 --> 00:23:24,980 What we see in polyketide biosynthesis 305 00:23:24,980 --> 00:23:26,570 is that those domains are optional. 306 00:23:30,620 --> 00:23:44,670 So one or more of these domains may be in a given module. 307 00:24:02,870 --> 00:24:06,170 So that's an overview, and then we'll 308 00:24:06,170 --> 00:24:12,020 look at an example of some domains and modules. 309 00:24:12,020 --> 00:24:17,300 So we're going to focus on type 1 polyketide synthases. 310 00:24:17,300 --> 00:24:20,300 And in these, what we're going to see 311 00:24:20,300 --> 00:24:25,880 is that catalytic and carrier protein domains are fused, 312 00:24:25,880 --> 00:24:29,750 and they're organized into what we'll term modules. 313 00:24:29,750 --> 00:24:35,240 So a module is defined as a group of domains that's 314 00:24:35,240 --> 00:24:42,000 responsible for activating, forming the carbon-carbon bonds 315 00:24:42,000 --> 00:24:43,640 and tailoring a monomer. 316 00:24:43,640 --> 00:24:47,780 So there is an individual module for every monomer 317 00:24:47,780 --> 00:24:49,670 within the growing chain. 318 00:24:49,670 --> 00:24:54,200 And the order of the modules in the polyketide synthase 319 00:24:54,200 --> 00:24:56,720 determines the functional group status, 320 00:24:56,720 --> 00:25:00,020 and that functional group status is determined by whether or not 321 00:25:00,020 --> 00:25:01,940 these optional domains are there. 322 00:25:01,940 --> 00:25:04,590 OK? 323 00:25:04,590 --> 00:25:07,410 How do we look for modules? 324 00:25:07,410 --> 00:25:11,730 The easiest way is to look for one of these thiolation or ACP 325 00:25:11,730 --> 00:25:12,930 domains. 326 00:25:12,930 --> 00:25:15,150 So each module has one of these. 327 00:25:15,150 --> 00:25:18,180 So you can count your number of T domains, 328 00:25:18,180 --> 00:25:21,180 and then you know, OK, there's 7T domains, 329 00:25:21,180 --> 00:25:25,020 so there's 7 monomers, for instance. 330 00:25:25,020 --> 00:25:28,890 So each Claisen condensation is a chain elongation and chain 331 00:25:28,890 --> 00:25:32,340 translocation event. 332 00:25:32,340 --> 00:25:34,260 Keep in mind, the starting monomer-- 333 00:25:34,260 --> 00:25:37,650 so whether that's acetyl-CoA or propionyl-CoA-- 334 00:25:37,650 --> 00:25:39,780 does not contain a CO2 group. 335 00:25:39,780 --> 00:25:42,810 So there's no decarboxylation of the starting monomer, 336 00:25:42,810 --> 00:25:45,270 but decarboxylation of malonyl-CoA 337 00:25:45,270 --> 00:25:47,220 occurs, like in fatty acid synthase, 338 00:25:47,220 --> 00:25:50,670 and if that's the case, it provides a C2 unit. 339 00:25:50,670 --> 00:25:53,160 And if methylmalonyl-CoA is the extender, 340 00:25:53,160 --> 00:25:55,620 this decarboxylation provides a C3 unit 341 00:25:55,620 --> 00:25:58,470 because of that methyl group. 342 00:25:58,470 --> 00:26:01,680 So key difference, as we just saw, 343 00:26:01,680 --> 00:26:05,160 in fatty acid biosynthesis, we have complete reduction of that 344 00:26:05,160 --> 00:26:08,040 beta-keto group in every elongation cycle 345 00:26:08,040 --> 00:26:10,290 because of these three tailoring domains-- 346 00:26:10,290 --> 00:26:12,820 the KR, DH, and ER. 347 00:26:12,820 --> 00:26:16,260 In PKS, what can happen is that reduction 348 00:26:16,260 --> 00:26:19,440 of this beta-keto group may not happen at all, 349 00:26:19,440 --> 00:26:23,280 or it may be incomplete in each elongation step. 350 00:26:23,280 --> 00:26:25,350 So what that means is that polyketides 351 00:26:25,350 --> 00:26:29,670 retain functional groups during chain elongation. 352 00:26:29,670 --> 00:26:32,160 And if you look back at some of the structures that 353 00:26:32,160 --> 00:26:34,710 were in the notes from last time, 354 00:26:34,710 --> 00:26:37,110 you'll see that, in terms of ketones, hydroxyls, 355 00:26:37,110 --> 00:26:39,450 double bonds, et cetera. 356 00:26:39,450 --> 00:26:41,130 And also, the other point to note 357 00:26:41,130 --> 00:26:43,770 is that there can be additional chemistry, 358 00:26:43,770 --> 00:26:48,580 and that these assembly lines where polyketide synthases, 359 00:26:48,580 --> 00:26:51,480 non-ribosomal peptide synthatases can contain what 360 00:26:51,480 --> 00:26:53,340 are called optional domains. 361 00:26:53,340 --> 00:26:54,960 So these are additional domains that 362 00:26:54,960 --> 00:26:58,020 are not required for formation of the carbon-carbon bond 363 00:26:58,020 --> 00:27:01,380 or amide bond in non-ribosomal peptide synthases. 364 00:27:01,380 --> 00:27:03,630 But they can do other chemistry there, so 365 00:27:03,630 --> 00:27:07,140 imagine a methyltransferase, for instance, or some cyclization 366 00:27:07,140 --> 00:27:08,920 domain. 367 00:27:08,920 --> 00:27:19,675 So how do we show these domains and modules? 368 00:27:22,460 --> 00:27:29,990 So typically, a given synthase is depicted from left to right 369 00:27:29,990 --> 00:27:38,520 in order of domain and bond-forming reactions here. 370 00:27:38,520 --> 00:27:39,790 So let's just take a look. 371 00:27:42,930 --> 00:27:58,030 So if we consider PKS domains and modules, 372 00:27:58,030 --> 00:28:26,610 we're just going to look at a pretend assembly line. 373 00:28:26,610 --> 00:28:27,200 OK? 374 00:28:27,200 --> 00:28:35,560 So this I'm defining here as an optional domain. 375 00:28:42,830 --> 00:28:46,540 So in this depiction, going from left to right, 376 00:28:46,540 --> 00:28:54,130 each one of these circles is a domain, so 377 00:28:54,130 --> 00:28:59,230 a polypeptide with a single enzymatic activity. 378 00:28:59,230 --> 00:29:02,560 Note that they're all basically touching one 379 00:29:02,560 --> 00:29:07,480 another which indicates in these types of notations 380 00:29:07,480 --> 00:29:09,425 that the polypeptide continues. 381 00:29:09,425 --> 00:29:11,050 It's not two different proteins, but we 382 00:29:11,050 --> 00:29:14,140 have one polypeptide here. 383 00:29:14,140 --> 00:29:17,440 I said that there's modules, and we can identify modules 384 00:29:17,440 --> 00:29:19,760 by counting T domains. 385 00:29:19,760 --> 00:29:23,320 So here, we have three T domains. 386 00:29:23,320 --> 00:29:25,015 So effectively there's three modules. 387 00:29:27,960 --> 00:29:40,050 So we have a module here, we have a module here, 388 00:29:40,050 --> 00:29:41,560 and we have a module here. 389 00:29:47,740 --> 00:29:48,390 What do we see? 390 00:29:48,390 --> 00:29:52,170 Two of these modules have a ketosynthase, 391 00:29:52,170 --> 00:29:54,690 so that's the domain that catalyzes the Claisen 392 00:29:54,690 --> 00:29:56,490 condensation. 393 00:29:56,490 --> 00:30:01,360 We have no ketosynthase here, in this first module. 394 00:30:01,360 --> 00:30:02,370 Why is that? 395 00:30:02,370 --> 00:30:04,330 We're all the way to the left. 396 00:30:04,330 --> 00:30:08,440 This is effectively our starter or loading module. 397 00:30:08,440 --> 00:30:12,870 So the propionyl-CoA or acetyl-CoA 398 00:30:12,870 --> 00:30:15,450 will be here, as we'll see, and there's nothing 399 00:30:15,450 --> 00:30:19,000 upstream to catalyze a condensate event with. 400 00:30:19,000 --> 00:30:22,290 So there's no KS domain in the starting 401 00:30:22,290 --> 00:30:24,700 module here or loading module. 402 00:30:24,700 --> 00:30:25,200 OK. 403 00:30:25,200 --> 00:30:33,245 So this is often called loading or starter. 404 00:30:38,790 --> 00:30:45,030 So if we think about these optional domains for a minute 405 00:30:45,030 --> 00:30:46,620 and think about how they work. 406 00:30:50,730 --> 00:31:01,660 If we go back to fatty acid synthase, and let's 407 00:31:01,660 --> 00:31:06,040 just imagine we have this species attached. 408 00:31:06,040 --> 00:31:12,890 We have the action of the KR, the dehydratase, 409 00:31:12,890 --> 00:31:20,830 and the ER to give us the fully-reduced species. 410 00:31:20,830 --> 00:31:24,970 Where here, we have a CH2 to group 411 00:31:24,970 --> 00:31:27,070 rather than the beta-ketone. 412 00:31:32,350 --> 00:31:36,760 So what happens in PKS in terms of 413 00:31:36,760 --> 00:31:38,620 the different optional domains? 414 00:31:38,620 --> 00:31:41,650 So we could have this and have full reduction. 415 00:31:41,650 --> 00:31:47,990 We can imagine maybe there's no enoyl reductase. 416 00:31:47,990 --> 00:31:50,800 So the module has the ketoreductase 417 00:31:50,800 --> 00:31:55,390 and the dehydratase but no enoyl reductase, and so as a result, 418 00:31:55,390 --> 00:32:02,200 this polyketide ends up with a double bond here. 419 00:32:02,200 --> 00:32:03,540 OK? 420 00:32:03,540 --> 00:32:11,460 What if we have nobody dehydratase, like this? 421 00:32:11,460 --> 00:32:11,960 OK. 422 00:32:11,960 --> 00:32:14,855 We just work backwards from the FAS cycle. 423 00:32:18,340 --> 00:32:21,620 We'd be left with this OH group at the beta position. 424 00:32:21,620 --> 00:32:22,120 Right? 425 00:32:22,120 --> 00:32:28,580 And if we have none of them, so no ketoreductase, dehydratase, 426 00:32:28,580 --> 00:32:35,740 or enoyl reductase, the beta-ketone 427 00:32:35,740 --> 00:32:40,120 will be retained, here. 428 00:32:44,130 --> 00:32:47,520 So what this also means is that you can just 429 00:32:47,520 --> 00:32:57,110 look at some polyketide and assess 430 00:32:57,110 --> 00:33:00,080 what the situation is from the standpoint 431 00:33:00,080 --> 00:33:02,360 of these optional domains. 432 00:33:02,360 --> 00:33:05,370 So let's just take an example. 433 00:33:05,370 --> 00:33:13,960 If we have three cycles of elongation, and let's 434 00:33:13,960 --> 00:33:25,720 imagine we had an acetyl-CoA starter plus three malonyl-CoA. 435 00:33:30,690 --> 00:33:32,100 So what do we end up with? 436 00:33:42,670 --> 00:33:44,440 Let's imagine our chain looks like this. 437 00:33:51,750 --> 00:33:53,700 What do we see? 438 00:33:53,700 --> 00:33:57,330 So two carbons are added during each elongation cycle 439 00:33:57,330 --> 00:34:03,570 to the chain here, and we can see those here, here, 440 00:34:03,570 --> 00:34:07,060 here, and here. 441 00:34:07,060 --> 00:34:08,280 OK? 442 00:34:08,280 --> 00:34:11,219 So a total of four C2 units, one from the starter 443 00:34:11,219 --> 00:34:14,070 and then three from these three extenders. 444 00:34:14,070 --> 00:34:17,310 And then we can look at what the functional group status is 445 00:34:17,310 --> 00:34:23,595 and say, OK, well here, we have no ketoreductase. 446 00:34:26,719 --> 00:34:30,560 And here, there was ketoreductase action, 447 00:34:30,560 --> 00:34:31,880 but there's no dehydratase. 448 00:34:37,090 --> 00:34:39,920 And here, what do we see? 449 00:34:39,920 --> 00:34:42,940 We see that there was a reduction of the beta-ketone 450 00:34:42,940 --> 00:34:45,040 and then the action of the dehydratase, 451 00:34:45,040 --> 00:34:52,060 but we're left at the alkene, so no enoyl reductase. 452 00:34:52,060 --> 00:34:52,560 Right? 453 00:34:52,560 --> 00:34:55,219 So just looking, you can begin to decipher 454 00:34:55,219 --> 00:34:58,260 in a given module what optional domains are there. 455 00:35:01,030 --> 00:35:06,270 So what we'll do is take a look at an actual PKS assembly line 456 00:35:06,270 --> 00:35:09,840 and then look at the chemistry happening on it here. 457 00:35:09,840 --> 00:35:11,565 These are just for your review. 458 00:35:14,260 --> 00:35:19,560 This is a polyketide synthase responsible for making 459 00:35:19,560 --> 00:35:24,010 this molecule here. 460 00:35:24,010 --> 00:35:30,210 So D-E-B or DEB is a 14-membered macrolactone. 461 00:35:30,210 --> 00:35:35,910 It's a precursor to the antibiotic erythromycin here, 462 00:35:35,910 --> 00:35:38,340 and this is the cartoon depiction 463 00:35:38,340 --> 00:35:42,900 of the polyketide synthase required for the biosynthesis 464 00:35:42,900 --> 00:35:45,960 of this molecule. 465 00:35:45,960 --> 00:35:52,020 So what do we see looking at this polyketide synthase? 466 00:35:52,020 --> 00:35:55,230 So it's more complicated than this one here, 467 00:35:55,230 --> 00:35:58,110 but the same principles apply. 468 00:35:58,110 --> 00:36:01,980 And what we'll see is that it's comprised of three proteins. 469 00:36:01,980 --> 00:36:06,210 There's seven modules, so one loading or starter module 470 00:36:06,210 --> 00:36:12,900 and six elongation modules, and there's a total of 28 domains. 471 00:36:12,900 --> 00:36:13,620 OK? 472 00:36:13,620 --> 00:36:16,920 And I said before, the placement and the identity 473 00:36:16,920 --> 00:36:20,340 of these domains dictates the identity of the growing chain. 474 00:36:20,340 --> 00:36:22,080 So let's take a look. 475 00:36:22,080 --> 00:36:26,100 So first, how do we know there's three proteins? 476 00:36:26,100 --> 00:36:28,410 We know that in this type of cartoon 477 00:36:28,410 --> 00:36:31,170 because we end up seeing some breaks 478 00:36:31,170 --> 00:36:33,370 between different domains. 479 00:36:33,370 --> 00:36:37,830 So here, for instance, the AT, the T, the KS, et cetera, 480 00:36:37,830 --> 00:36:40,680 they're all attached to one another in the cartoon. 481 00:36:40,680 --> 00:36:42,870 That means it's all one polypeptide chain, 482 00:36:42,870 --> 00:36:44,730 but this one polypeptide chain has 483 00:36:44,730 --> 00:36:47,490 many different enzymatic activities in it, 484 00:36:47,490 --> 00:36:49,740 because it has different domains. 485 00:36:49,740 --> 00:36:50,790 When we see a break-- 486 00:36:50,790 --> 00:36:54,540 so for instance here this T domain and this KS domain 487 00:36:54,540 --> 00:36:56,250 are not touching one another. 488 00:36:56,250 --> 00:36:59,410 That means we have two separate proteins. 489 00:36:59,410 --> 00:37:03,540 So this T domain is at the terminus of DEBS 1, 490 00:37:03,540 --> 00:37:06,880 and DEBS 2 begins with this ketosynthase. 491 00:37:06,880 --> 00:37:07,650 OK? 492 00:37:07,650 --> 00:37:11,070 Likewise, we have a break here, between the T domain 493 00:37:11,070 --> 00:37:12,930 and this ketosynthase. 494 00:37:12,930 --> 00:37:16,642 So three proteins make up this assembly line, 495 00:37:16,642 --> 00:37:18,600 and so when thinking about this, these proteins 496 00:37:18,600 --> 00:37:21,360 are going to have to interact with each other in one 497 00:37:21,360 --> 00:37:22,450 way or another. 498 00:37:22,450 --> 00:37:25,560 And so there's a lot of dynamics in protein-protein interactions 499 00:37:25,560 --> 00:37:27,570 happening here. 500 00:37:27,570 --> 00:37:30,090 How do we know there's seven modules? 501 00:37:30,090 --> 00:37:36,300 And remember each module is responsible for one monomer 502 00:37:36,300 --> 00:37:37,110 unit. 503 00:37:37,110 --> 00:37:41,558 We count the T domains, so we have one, two, three, four, 504 00:37:41,558 --> 00:37:44,910 five, six, seven T domains. 505 00:37:44,910 --> 00:37:46,700 So like the acyl carrier proteins 506 00:37:46,700 --> 00:37:49,680 of fatty acid synthase, these T domains 507 00:37:49,680 --> 00:37:53,400 will be post-translationally modified with a PPant arm. 508 00:37:53,400 --> 00:37:55,890 And that PPant arm will be loaded 509 00:37:55,890 --> 00:38:00,690 with the acetyl-CoA or methylmalonyl-CoA or 510 00:38:00,690 --> 00:38:03,960 malonyl-CoA monomers. 511 00:38:03,960 --> 00:38:05,500 We have a loading module. 512 00:38:05,500 --> 00:38:07,890 So the loading module has no ketosynthase, 513 00:38:07,890 --> 00:38:10,860 because there's nothing upstream over here 514 00:38:10,860 --> 00:38:14,000 for catalyzing a carbon-carbon bond formation event. 515 00:38:14,000 --> 00:38:16,710 And then we see modules one through six, 516 00:38:16,710 --> 00:38:21,090 so sometimes the loading module is module zero. 517 00:38:21,090 --> 00:38:25,050 We see that each one has a ketosynthase, 518 00:38:25,050 --> 00:38:27,360 so there'll be carbon-carbon bond formation 519 00:38:27,360 --> 00:38:29,550 going along this assembly line. 520 00:38:29,550 --> 00:38:32,700 And we see that the optional domains vary. 521 00:38:32,700 --> 00:38:36,390 So for instance, module one has a ketoreductase 522 00:38:36,390 --> 00:38:38,580 as does module two. 523 00:38:38,580 --> 00:38:39,720 Look at module four. 524 00:38:39,720 --> 00:38:43,140 We see all three domains required 525 00:38:43,140 --> 00:38:48,270 for complete processing of that beta-keto group here. 526 00:38:48,270 --> 00:38:52,570 Here, only a ketoreductase, and here only a ketoreductase. 527 00:38:52,570 --> 00:38:53,070 OK? 528 00:38:53,070 --> 00:38:54,880 So just looking at this, you can say, 529 00:38:54,880 --> 00:38:58,680 OK well, we'll have an OH group here, here. 530 00:38:58,680 --> 00:39:00,870 Here we have complete processing. 531 00:39:00,870 --> 00:39:01,830 Just ignore this. 532 00:39:01,830 --> 00:39:06,310 It's in lower case, because it's a non-functional reductase 533 00:39:06,310 --> 00:39:07,200 domain. 534 00:39:07,200 --> 00:39:12,000 It's not operating as annotated here. 535 00:39:12,000 --> 00:39:15,310 So what happens? 536 00:39:15,310 --> 00:39:18,300 So again, there's post-translational modification 537 00:39:18,300 --> 00:39:21,530 of this T domain, so it has a serine. 538 00:39:21,530 --> 00:39:26,160 The serine gets modified with the PPant arm, as shown here, 539 00:39:26,160 --> 00:39:28,920 and we use that squiggle depiction, 540 00:39:28,920 --> 00:39:33,630 as I showed for the acyl carrier protein of FAS. 541 00:39:33,630 --> 00:39:36,510 So post-translational modification of these T domains 542 00:39:36,510 --> 00:39:39,390 has to happen before any of the monomers 543 00:39:39,390 --> 00:39:41,910 are loaded onto this assembly line. 544 00:39:41,910 --> 00:39:47,580 And these PPant arms allow us to use bioesters as the linkages 545 00:39:47,580 --> 00:39:50,470 and through the chemistry I showed earlier. 546 00:39:50,470 --> 00:39:56,100 So here, what we're seeing in this cartoon, going from here, 547 00:39:56,100 --> 00:39:58,560 this indicates that the T domains are not 548 00:39:58,560 --> 00:40:00,940 post-translationally modified. 549 00:40:00,940 --> 00:40:04,080 And here, we see the assembly line 550 00:40:04,080 --> 00:40:08,460 after action of some [? phosphopentyltransferase ?] 551 00:40:08,460 --> 00:40:10,850 loading these arms. 552 00:40:10,850 --> 00:40:11,430 OK? 553 00:40:11,430 --> 00:40:16,380 So each T domain gets post-translationally modified. 554 00:40:16,380 --> 00:40:17,910 What happens next? 555 00:40:17,910 --> 00:40:20,220 We have loading of monomers. 556 00:40:20,220 --> 00:40:23,040 And we'll look at module zero and one on the board 557 00:40:23,040 --> 00:40:26,220 and then look at how the whole assembly line goes. 558 00:40:30,945 --> 00:40:32,590 AUDIENCE: Do you ever get selected 559 00:40:32,590 --> 00:40:36,260 post-translational modification of the T domains 560 00:40:36,260 --> 00:40:39,290 and if so, does that facilitate different modules being 561 00:40:39,290 --> 00:40:40,950 like on or off, so to speak? 562 00:40:40,950 --> 00:40:42,200 ELIZABETH NOLAN: I don't know. 563 00:40:42,200 --> 00:40:45,050 I don't know in terms of the kinetics, 564 00:40:45,050 --> 00:40:48,860 and say, does one T domain get loaded by a PPTase 565 00:40:48,860 --> 00:40:52,430 before the other? 566 00:40:52,430 --> 00:40:56,090 These enzymes are very complex, and there's 567 00:40:56,090 --> 00:40:58,040 a lot we don't know. 568 00:40:58,040 --> 00:41:01,370 But that would be interesting, if it's the case. 569 00:41:01,370 --> 00:41:04,784 I wouldn't rule it out, but I just don't know. 570 00:41:04,784 --> 00:41:10,700 One thing to point out too, these assembly lines are huge. 571 00:41:10,700 --> 00:41:14,690 So this is something we'll talk about more the next time, 572 00:41:14,690 --> 00:41:18,690 as we begin to discuss how do you experimentally study them? 573 00:41:18,690 --> 00:41:23,030 But some are the size of the ribosome for the biosynthesis 574 00:41:23,030 --> 00:41:25,200 of one natural product. 575 00:41:25,200 --> 00:41:27,050 And what that means, from the standpoint 576 00:41:27,050 --> 00:41:29,420 of in vitro characterization, is that often 577 00:41:29,420 --> 00:41:33,350 you just can't express a whole assembly line, 578 00:41:33,350 --> 00:41:36,800 let alone say one protein that has a few modules. 579 00:41:36,800 --> 00:41:40,790 So often, what people will do is individually express domains 580 00:41:40,790 --> 00:41:43,910 or dye domains and study the reactions 581 00:41:43,910 --> 00:41:46,560 they catalyze in their chemistry there. 582 00:41:46,560 --> 00:41:49,640 And so it would be very difficult even 583 00:41:49,640 --> 00:41:52,850 to test that in terms of in vitro. 584 00:41:52,850 --> 00:41:56,470 Is there an ordering to how the T domains are loaded? 585 00:41:56,470 --> 00:41:58,220 And then there's question too, do you even 586 00:41:58,220 --> 00:42:01,310 know what the dedicated PPTase is? 587 00:42:01,310 --> 00:42:04,310 So there's some tricks that are done on the bench top 588 00:42:04,310 --> 00:42:07,640 to get around not knowing that, which we'll talk about later. 589 00:42:07,640 --> 00:42:22,210 So back to this assembly line to make DEB. 590 00:42:22,210 --> 00:42:25,180 So we're just going to go over the loading module 591 00:42:25,180 --> 00:42:30,160 and module 1 and look at a Claisen condensation catalyzed 592 00:42:30,160 --> 00:42:31,420 by the KS. 593 00:42:31,420 --> 00:42:36,310 And this chemistry pertains to the various other modules 594 00:42:36,310 --> 00:42:37,940 and other PKS. 595 00:42:37,940 --> 00:42:48,600 So we have our AT domain and our thiolation domain of module 0, 596 00:42:48,600 --> 00:42:57,950 and then we have the ketosynthase, the AT domain, 597 00:42:57,950 --> 00:43:06,950 the ketoreductase, and the T domain of module 1. 598 00:43:06,950 --> 00:43:07,450 OK. 599 00:43:07,450 --> 00:43:09,610 I'm drawing these a little up and down just 600 00:43:09,610 --> 00:43:11,710 to make it easier to show the chemistry. 601 00:43:11,710 --> 00:43:13,180 So sometimes you see them straight, 602 00:43:13,180 --> 00:43:16,870 sometimes moved around here, but it's all the same. 603 00:43:16,870 --> 00:43:24,760 So we have these PPant arms on the two T domains. 604 00:43:24,760 --> 00:43:28,270 So what happens now, after these have been post-translationally 605 00:43:28,270 --> 00:43:29,650 modified? 606 00:43:29,650 --> 00:43:32,140 We need the action of the AT domains 607 00:43:32,140 --> 00:43:36,160 to load the monomers onto the PPant arms 608 00:43:36,160 --> 00:43:42,800 here, so action of the AT domain. 609 00:44:10,780 --> 00:44:12,040 So what do we end up with? 610 00:44:12,040 --> 00:44:18,900 In this case, the starter is a propionyl-CoA, 611 00:44:18,900 --> 00:44:20,430 so we can see that here. 612 00:44:30,530 --> 00:44:36,430 And we have a methylmalonyl-CoA as the extender, that 613 00:44:36,430 --> 00:44:46,420 gets loaded, and I'm going to draw the cysteine thiolate 614 00:44:46,420 --> 00:44:49,870 of the ketosynthase here. 615 00:44:49,870 --> 00:44:51,430 So what happens next? 616 00:44:54,820 --> 00:44:59,350 We need to have decarboxylation of the methylmalonyl-CoA 617 00:44:59,350 --> 00:45:02,440 monomer to give us a C3 unit. 618 00:45:02,440 --> 00:45:05,140 And it's C3 because of this methyl group, 619 00:45:05,140 --> 00:45:07,840 but the growing chain will grow by two carbons. 620 00:45:07,840 --> 00:45:12,070 And then we need to have transfer of this starter 621 00:45:12,070 --> 00:45:13,630 to the ketosynthase. 622 00:45:13,630 --> 00:45:18,220 So the ketosynthase is involved in covalent catalysis here. 623 00:45:18,220 --> 00:45:24,190 So what happens, we can imagine here, we have attack, 624 00:45:24,190 --> 00:45:27,640 and then here, we're going to have the decarboxylation. 625 00:45:55,730 --> 00:46:03,860 We have chain transfer to the ketosynthase, 626 00:46:03,860 --> 00:46:12,330 and here, decarboxylation leaves us this species. 627 00:46:12,330 --> 00:46:12,830 OK? 628 00:46:51,810 --> 00:46:53,340 OK. 629 00:46:53,340 --> 00:46:54,630 So now, what happens? 630 00:46:54,630 --> 00:46:59,940 Now, the assembly's set up for the Claisen condensation 631 00:46:59,940 --> 00:47:05,530 to occur which is catalyzed by the ketosynthase. 632 00:47:05,530 --> 00:47:06,030 Right? 633 00:47:06,030 --> 00:47:08,080 So what will happen here? 634 00:47:08,080 --> 00:47:17,490 You can imagine that, and as a result, where do we end up? 635 00:47:17,490 --> 00:47:18,840 I'll just draw it down here. 636 00:47:49,010 --> 00:47:50,120 And what else do we have? 637 00:47:50,120 --> 00:47:55,760 We have a ketoreductase. 638 00:47:55,760 --> 00:47:59,900 So this ketoreductase will act on the monomer 639 00:47:59,900 --> 00:48:03,110 of the upstream unit, and that's how it always is. 640 00:48:03,110 --> 00:48:07,130 So if there's optional domains in module 1, 641 00:48:07,130 --> 00:48:10,490 they act on the monomer from module 0. 642 00:48:10,490 --> 00:48:13,160 If there's optional domains in module 2, 643 00:48:13,160 --> 00:48:15,740 they'll act on the monomer for module 1. 644 00:48:15,740 --> 00:48:16,400 OK? 645 00:48:16,400 --> 00:48:20,330 So we see here now we have reduction 646 00:48:20,330 --> 00:48:26,080 of the ketone from module 1 to here via the ketoreductase. 647 00:48:26,080 --> 00:48:26,580 OK? 648 00:48:48,960 --> 00:48:58,530 So if we take a look at what's on the PowerPoint 649 00:48:58,530 --> 00:49:03,210 here, what we're seeing is one depiction of this assembly line 650 00:49:03,210 --> 00:49:07,720 to make DEB indicating the growing chain. 651 00:49:07,720 --> 00:49:08,220 OK? 652 00:49:08,220 --> 00:49:11,550 So as we walk through each module, 653 00:49:11,550 --> 00:49:14,850 we see an additional monomer attached. 654 00:49:14,850 --> 00:49:16,650 So the chain elongates, and then you 655 00:49:16,650 --> 00:49:21,690 can track what's happening to the ketone group 656 00:49:21,690 --> 00:49:25,080 of the upstream monomer on the basis of the optional domains 657 00:49:25,080 --> 00:49:27,300 here. 658 00:49:27,300 --> 00:49:30,930 If we look in this one, which I like this one because they 659 00:49:30,930 --> 00:49:32,250 color code. 660 00:49:32,250 --> 00:49:36,730 So they color code the different modules along with the monomer, 661 00:49:36,730 --> 00:49:39,640 and so it's pretty easy to trace what's happening. 662 00:49:39,640 --> 00:49:44,080 So for instance, here we have the loading module, 663 00:49:44,080 --> 00:49:46,980 and we have the starter unit in red. 664 00:49:46,980 --> 00:49:50,400 And here we see that it's been reduced by the ketoreductase 665 00:49:50,400 --> 00:49:52,650 of the upstream blue module. 666 00:49:52,650 --> 00:49:55,590 Here, we have the green module, here is its monomer, 667 00:49:55,590 --> 00:49:59,670 and we see its ketoreductase acted on the blue monomer 668 00:49:59,670 --> 00:50:02,040 from module 1, et cetera here. 669 00:50:02,040 --> 00:50:05,730 So I encourage you all to just very systematically work 670 00:50:05,730 --> 00:50:10,260 through the assembly lines that are provided in these notes, 671 00:50:10,260 --> 00:50:13,990 and it's the same type of chemistry over and over again. 672 00:50:13,990 --> 00:50:15,420 And if you learn the patterns, it 673 00:50:15,420 --> 00:50:18,540 ends up being quite easy to work through, at least 674 00:50:18,540 --> 00:50:19,990 the simple assembly line. 675 00:50:19,990 --> 00:50:23,470 So as you can imagine, complexity increases, 676 00:50:23,470 --> 00:50:26,760 and we'll look at some examples of more complex ones as well. 677 00:50:26,760 --> 00:50:29,040 So where we'll start next time with this 678 00:50:29,040 --> 00:50:33,720 is just briefly looking at chain release by the thioesterase. 679 00:50:33,720 --> 00:50:37,320 And then we'll do an overview of non-ribosomal peptide 680 00:50:37,320 --> 00:50:42,370 biosynthesis logic and then look at some example assembly lines. 681 00:50:42,370 --> 00:50:46,620 So we have the exams to give back. 682 00:50:46,620 --> 00:50:48,840 I'll just say a few things. 683 00:50:48,840 --> 00:50:56,090 So the average was around a 68, plus or minus 10, 11, 684 00:50:56,090 --> 00:50:58,530 12 for the standard deviation. 685 00:50:58,530 --> 00:51:01,560 I'd say, if you were in the mid 70s and above, 686 00:51:01,560 --> 00:51:03,390 you did really well. 687 00:51:03,390 --> 00:51:07,500 If you're into the low 60s, that is OK, 688 00:51:07,500 --> 00:51:12,180 but we'd really like things to improve for the next one. 689 00:51:12,180 --> 00:51:17,205 In terms of the exam and just some feedback-- and I'll 690 00:51:17,205 --> 00:51:20,160 put feedback as well in the key which will be posted 691 00:51:20,160 --> 00:51:21,990 later today or early tomorrow. 692 00:51:21,990 --> 00:51:25,880 There wasn't one question that say the whole class bombed, 693 00:51:25,880 --> 00:51:27,180 so that's good. 694 00:51:27,180 --> 00:51:29,552 There were a few things for just general improvement, 695 00:51:29,552 --> 00:51:32,010 and I want to bring this up, so you can also think about it 696 00:51:32,010 --> 00:51:33,900 in terms of problem sets. 697 00:51:33,900 --> 00:51:37,420 One involves being quantitative. 698 00:51:37,420 --> 00:51:41,080 So there's certainly qualitative trends and data, 699 00:51:41,080 --> 00:51:44,340 but there's also quantitative information there, 700 00:51:44,340 --> 00:51:46,830 and that can be important to look at. 701 00:51:46,830 --> 00:51:51,030 And one example I'll give of that involved question one. 702 00:51:51,030 --> 00:51:55,080 If you recall, there was an analysis of GDP hydrolysis 703 00:51:55,080 --> 00:51:58,260 and an analysis of peptide bond formation. 704 00:51:58,260 --> 00:52:03,360 And quantitative analysis of the peptide bond formation 705 00:52:03,360 --> 00:52:08,850 experiments will show that all of the lysyl-tRNAs 706 00:52:08,850 --> 00:52:14,130 were used up in the case of the codon that was AAA. 707 00:52:14,130 --> 00:52:16,800 Whereas, some of those tRNAs were not 708 00:52:16,800 --> 00:52:22,710 used up when the codon contained that 6-methyl-A 709 00:52:22,710 --> 00:52:24,370 in position one. 710 00:52:24,370 --> 00:52:24,870 Right? 711 00:52:24,870 --> 00:52:28,200 And if you linked that back to the kinetic model 712 00:52:28,200 --> 00:52:30,240 along with the other data, what that indicates 713 00:52:30,240 --> 00:52:31,840 is that proofreading is going on. 714 00:52:31,840 --> 00:52:32,340 Right? 715 00:52:32,340 --> 00:52:37,050 Some of those tRNAs are being rejected from the ribosome 716 00:52:37,050 --> 00:52:37,740 there. 717 00:52:37,740 --> 00:52:41,160 So that was one place where quantitiation, a fair number 718 00:52:41,160 --> 00:52:43,570 of you missed that. 719 00:52:43,570 --> 00:52:45,300 And another thing I just want to stress 720 00:52:45,300 --> 00:52:49,740 is to make sure you answered the question being asked. 721 00:52:49,740 --> 00:52:54,300 And where an example of that came up was in question one 722 00:52:54,300 --> 00:52:58,140 with the final question asking about relating the data back 723 00:52:58,140 --> 00:52:59,997 to the kinetic model. 724 00:52:59,997 --> 00:53:01,830 And so if a question asks that you really do 725 00:53:01,830 --> 00:53:04,470 need to go back to the model which was in the appendix 726 00:53:04,470 --> 00:53:05,970 and think about that. 727 00:53:05,970 --> 00:53:08,490 So many of you gave some very interesting answers 728 00:53:08,490 --> 00:53:12,495 and presented hypotheses about perhaps the 6-methyl-A 729 00:53:12,495 --> 00:53:15,225 is involved in regulation and controlling 730 00:53:15,225 --> 00:53:17,780 like the timing of translation. 731 00:53:17,780 --> 00:53:20,680 And that's terrific and interesting to think about, 732 00:53:20,680 --> 00:53:22,660 but it wasn't the answer to the question. 733 00:53:22,660 --> 00:53:23,160 Right? 734 00:53:23,160 --> 00:53:26,130 Which was to go beyond the conclusions 735 00:53:26,130 --> 00:53:29,100 from the experiments with GTP hydrolysis 736 00:53:29,100 --> 00:53:32,910 and formation of that dipeptide, and ask 737 00:53:32,910 --> 00:53:35,580 how can we conceptualize this from the standpoint 738 00:53:35,580 --> 00:53:37,950 of the model we studied in class? 739 00:53:37,950 --> 00:53:41,280 And then just the third point I'll make 740 00:53:41,280 --> 00:53:49,080 is related to question two and specifically to GroEL. 741 00:53:49,080 --> 00:53:52,560 But the more general thing is that if we 742 00:53:52,560 --> 00:53:55,110 learn about a system in class, unless there's 743 00:53:55,110 --> 00:53:57,150 compelling data presented in a question 744 00:53:57,150 --> 00:54:00,263 to suggest the model is something other than what we 745 00:54:00,263 --> 00:54:02,430 learned or its behavior is something other than what 746 00:54:02,430 --> 00:54:05,550 we learned, stick with what you know. 747 00:54:05,550 --> 00:54:10,530 So in the use of GroEL, the idea in that experiment 748 00:54:10,530 --> 00:54:14,640 was that, if you recall, this question was looking at these J 749 00:54:14,640 --> 00:54:17,730 proteins and asking, how do J proteins 750 00:54:17,730 --> 00:54:20,050 facilitate disaggregation? 751 00:54:20,050 --> 00:54:20,550 Right? 752 00:54:20,550 --> 00:54:26,070 And so a GroEL trap was used that cannot hydrolyze ATP, 753 00:54:26,070 --> 00:54:33,240 which means it's not active at folding any polypeptide. 754 00:54:33,240 --> 00:54:37,170 But the idea there is that these J proteins end up 755 00:54:37,170 --> 00:54:40,320 allowing monomers to come out of the aggregate, 756 00:54:40,320 --> 00:54:43,320 and then GroEL can trap and unfold the monomer 757 00:54:43,320 --> 00:54:46,290 to prevent reactivation. 758 00:54:46,290 --> 00:54:49,200 And so a number of people came to the conclusion 759 00:54:49,200 --> 00:54:52,500 that GroEL was binding that aggregate somehow 760 00:54:52,500 --> 00:54:53,580 in its chamber. 761 00:54:53,580 --> 00:54:55,200 And what we learned about GroEL is 762 00:54:55,200 --> 00:54:59,060 that its chamber can't house a protein over 60 kilodaltons. 763 00:54:59,060 --> 00:54:59,560 Right? 764 00:54:59,560 --> 00:55:01,800 We saw that in terms of the in vitro assays 765 00:55:01,800 --> 00:55:05,010 that were done looking at what its native substrates are. 766 00:55:05,010 --> 00:55:05,510 Right? 767 00:55:05,510 --> 00:55:07,950 So always go back to what you know, and then you 768 00:55:07,950 --> 00:55:10,080 need to ask yourselves, are the data 769 00:55:10,080 --> 00:55:12,950 suggesting some other behavior? 770 00:55:12,950 --> 00:55:15,050 And if that were the case, like what 771 00:55:15,050 --> 00:55:18,070 is your analysis of those data there? 772 00:55:18,070 --> 00:55:22,530 So please, even if you did really well, look at the key 773 00:55:22,530 --> 00:55:24,107 and see what the key has to say. 774 00:55:24,107 --> 00:55:26,690 And if you have questions, you can make an appointment with me 775 00:55:26,690 --> 00:55:30,970 or come to office hours or discuss with Shiva there. 776 00:55:30,970 --> 00:55:32,520 OK?