Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and pol yketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reactio n is generated by decarboxylation of a malonyl or methylmalonyl moiety; nor mally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for t he ensuing condensation reaction. Replacement of the cysteine nucleophile ( Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multif unctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by mor e than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ke toacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wi ld type and is similar to that exhibited by the wild-type beta-ketoacyl syn thase in the presence of an acetyl primer. These results, together with mod eling studies of the Cys --> Gin mutant from the crystal structure of the E scherichia coli beta-ketoacyl synthase II enzyme, suggest that the side cha in carbonyl group of the Gln161 can mimic the carbonyl of the acyl moiety i n the acyl-enzyme intermediate so that the mutant adopts a conformation ana logous to that of the acyl-enzyme intermediate. Catalysis of the decarboxyl ation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA est er to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gin beta-ketoacyl synthases found in the loading domain s of some modular polyketide synthases likely is to act as malonyl, or meth ylmalonyl, decarboxylases that provide a source of primer for the chain ext ension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.