Substrate structure–activity relationships guide rational engineering of modular polyketide synthase ketoreductases

Bailey, Constance B.; Pasman, Marjolein; Keatinge‐Clay, Adrian T.

doi:10.1039/c5cc07315d

Cited by 37 publications

(61 citation statements)

References 30 publications

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“…23 Activity assays established that the k cat / K m for reduction by EryKR6-G324T/L333H was essentially the same as that of wild-type EryKR6, while TylKR1-Q377H showed a ~16-fold decrease in k cat / K m compared to the parent TylKR1 (Table S5). 25 …”

Section: Resultsmentioning

confidence: 99%

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

et al. 2017

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The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly-generated, transiently-formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

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Section: Resultsmentioning

confidence: 99%

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

et al. 2017

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“…4B). The Felkin-Ahn rule predicts the reactions catalyzed by A2-type KRs are more favorable compared with the reactions catalyzed byA1-type KRs (Bailey et al, 2016; Mengel and Reiser, 1999). Therefore, the generation of a (2L, 3L)-product by the G355T/Q364H mutant is likely due to a decreased capability for stereocontrol.…”

Section: Discussionmentioning

confidence: 99%

Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase

Liu

Yuan

et al. 2018

Journal of Structural Biology

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Ketoreductase (KR) domains of modular polyketide synthases (PKSs) control the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates. To understand the molecular basis of stereocontrol exerted by KRs, the crystal structure of a KR from the second module of the amphotericin PKS (AmpKR2) complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine was solved. This first ternary structure provides direct evidence to the hypothesis that a substrate enters into the active site of an A-type KR from the side opposite the coenzyme to generate an L-hydroxyl substituent. A comparison with the ternary complex of a G355T/Q364H mutant sheds light on the structural basis for stereospecificity toward the substrate C2 methyl substituent. Functional assays suggest the pantetheine handle shows obvious influence on the catalytic efficiency and the stereochemical outcome. Together, these findings extend our current understanding of the stereo-chemical control of PKS KR domains.

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“…38–40 Intriguingly, neither extensive sequence alignments nor crystal structures of both epimerase-active and epimerase-inactive KR domains have so far revealed any plausible acidic or basic amino acid residues suitably positioned within the active sites to mediate deprotonation or reprotonation at C-2 of the epimerizable substrate. 8–13,38–40 We have previously ruled out any role for the NADPH cofactor in the epimerization reaction itself. 18 Why some KR domains have epimerase activity and some do not is an essential question whose answer remains to be established.…”

Section: Discussionmentioning

confidence: 99%

Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues

et al. 2016

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The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR30 and PicKR30 as well as redox-inactive mutants of EryKR1, were incubated with [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-2) and 0.05 equiv of NADP+ in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by LC-MS-MS with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.

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Substrate structure–activity relationships guide rational engineering of modular polyketide synthase ketoreductases

Cited by 37 publications

References 30 publications

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase

Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues

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