Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases.
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 an ~10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed an ~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.
Departments of Chemistry and Chemical Engineering, Stanford University, Stanford, CA, USA.
Matthew P
MP
Ostrowski
Department of Chemistry, Brown University, Providence, RI, USA.
David E
DE
Cane
Departments of Chemistry and Chemical Engineering, Stanford University, Stanford, CA, USA.
Chaitan
C
Khosla
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D048630:Polyketide Synthases; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase