Stereospecific Acyl Transfers on the Erythromycin-Producing Polyketide Synthase

Marsden, Andrew F. A.; Caffrey, Patrick; Aparicio, Jesús F.; Loughran, Mark S.; Staunton, James; Leadlay, Peter F.

doi:10.1126/science.8278811

Cited by 169 publications

(145 citation statements)

References 24 publications

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“…The malonyl-S-ACP product is apparently not stable under the assay condition, the malonyl group of which undergoes hydrolysis in the absence of chain elongation. Analogous results have been found for both the 6-deoxyerythronolide B synthase (24) and fatty acid synthase (26). In contrast, LnmG labeling appeared to be constant (Fig.…”

Section: Lnmg Loads Malonyl Coa In Trans To All Six Pks Modules Of Lnsupporting

confidence: 59%

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Tang

Shen

2003

Proc. Natl. Acad. Sci. U.S.A.

242

258

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Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a ␤-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to ''AT-less'' multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.

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Section: Lnmg Loads Malonyl Coa In Trans To All Six Pks Modules Of Lnsupporting

confidence: 59%

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Tang

Shen

2003

Proc. Natl. Acad. Sci. U.S.A.

242

258

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“…28 At this scale, each enzyme incubation can be carried out in a convenient volume of 500 μL, using [10][11][12][13][14][15][16][17][18][19][20] …”

Section: Gc-ms Analysis Of Triketide Lactone Diastereomersmentioning

confidence: 99%

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Castonguay

Chen

et al. 2007

J. Am. Chem. Soc.

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Abstract6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS) -acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base-or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-mass spectrometry technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACPbound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.Modular polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of polyketides metabolites that exhibit a wide range of important biological properties, including antibacterial, antifungal, anticancer, and immunosuppressive activity. 1 They catalyze repetitive Claisen-like condensations between methylmalonyl or malonyl thioester building blocks and the growing polyketide acyl thioesters. Using an assembly line of active sites, each module is responsible for one cycle of polyketide chain elongation and *To whom correspondence should be addressed. E-mail: David_Cane@brown.edu. (TE) domain, located at the C-terminus of the furthest downstream module, catalyzes release and concomitant cyclization of the mature, full-length macrocyclic polyketide. NIH Public AccessThe 6-deoxyerythronolide synthase (DEBS) from Saccharopolyspora erythraea, which is by far the most thoroughly studied modular PKS, is responsib...

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“…Ery 0 and Ave 0 indicate AT domains that load the starter unit in the erythromycin [12] and avermectin PKSs respectively. required for loading of propionyl-CoA and the other six all incorporate methylmalonyl-CoA [5,12], localised the divergent substrate-specific sequence motifs to a relatively small stretch of 20 amino acids, N-terminal of the catalytic serine residue found in the consensus sequence GXSXG common to all these enzymes (Fig. 1).…”

Section: Starter Atsmentioning

confidence: 99%

“…It was subsequently shown that in fact all six methylmalonyltransferase domains of the erythromycin-producing polyketide synthase possess the same substrate stereospecificity, for (2S)-methylmalonyl-CoA, and the overall outcome must be governed by other enzyme components of the synthase [5].…”

Section: Introductionmentioning

confidence: 99%

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

et al. 1995

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The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences are sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate.

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Stereospecific Acyl Transfers on the Erythromycin-Producing Polyketide Synthase

Cited by 169 publications

References 24 publications

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

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