Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases

Abstract

Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules.

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

“…A., T. R., and B. L. designed the study. M. K. purified the proteins, constructed TE-knockout mutants, and carried out the NADPH consumption, LC-MS analysis, and 14 C radiolabeling experiments. J.…”

“…Following destaining with water for 5 min, the gel was mounted on a filter paper and dried in vacuo for 2 h using a Bio-Rad 543 gel dryer. The dried gel was imaged for 1 h lane by lane to quantify 14 C on individual protein bands using a Rita Star TLC Analyzer (Raytest). Peaks were integrated to quantify the radiolabel bound to each protein.…”

“…This critical distinction has provided a critical mechanistic framework for factoring the contribution of imperfect intermodular ACP-KS interactions to the turnover efficiency of chimeric assembly line PKSs. We have also established that the KR domain of DEBS M2, which generates the enantiomeric (2R, 3S)-2-methyl-3-hydroxy diketide diastereomer of the natural (2S, 3R) product of DEBS M1, has comparable specificity for the ACP domain of DEBS M1 compared with its natural ACP2 substrate (14). This finding has now allowed us to investigate the influence of the recognition of substrate stereochemistry by an acceptor KS on the overall turnover by otherwise identical chimeric PKSs.…”

The Ketosynthase Domain Constrains the Design of Polyketide Synthases

“…With the aim of additionally analyzing the contribution of ACP:KS recognition and substrate:KS interaction on the turnover of KS mutants, we generated alternative M1 donor modules that would allow us to probe the remaining ACP-substrate combinations ( Figure 4&5). Based on the observations that KR domains can efficiently turn over with a variety of chimeric ACPs, 39 and that chain elongation at a chimeric ACP:KS interface in a fusion protein is not rate-limiting, 22 we generated 6 alternative M1 donors by permutating KR1 and ACP1. The exchange of ACP1 with ACP2 and ACP5, the native upstream ACPs of M3/M6, generated donor modules carrying NDK on the respective ACP ( Figure 4A, donor 2 & Figure 5A, donor 5).…”

Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects

“…These advances in re-engineering the stereochemical outcomes of the KR domain has been complemented by investigations into its ACP specificity. The interaction landscape of four reducing KRs from DEBS and five ACPs from DEBS was assessed in a simple in vitro assay [ 87 ]. The outcomes showed that KRs often exhibit a modest preference for their cognate ACPs by a factor of 10- to 50-fold, although this rule was violated by KR6, which preferred ACP5 instead.…”