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Kinetic and Mechanistic Analysis of the Malonyl CoA:ACP Transacylase from Streptomyces coelicolor Indicates a Single Catalytically Competent Serine Nucleophile at the Active Site

2002; American Chemical Society; Volume: 41; Issue: 5 Linguagem: Inglês

10.1021/bi012001p

1943-295X

Anna E. Szafranska, Timothy S. Hitchman, Russell J. Cox, John Crosby, Thomas J. Simpson,

Chemical Synthesis and Analysis

The source of malonyl groups for polyketide and fatty acid biosynthesis is malonyl CoA. During fatty acid and polyketide biosynthesis, malonyl groups are normally transferred to the acyl carrier protein (ACP) component of the synthase by a malonyl CoA:holo-ACP transacylase (MCAT) enzyme. The fatty acid synthase (FAS) malonyl CoA:ACP transacylase from Streptomyces coelicolor was expressed in Escherichia coli as a hexahistidine-tagged (His6) fusion protein in high yield. The His6-MCAT was purified to homogeneity using standard techniques, and kinetic analysis of the malonylation of S. coelicolor FAS holo-ACP, catalyzed by His6-MCAT, gave values of 73 (ACP) and 60 μM (malonyl CoA). A catalytic constant of 450 s-1 and specificity constants / of 6.2 (ACP) and 7.5 μM-1 s-1 (malonyl CoA) were measured. Malonyl transfer to the E. coli FAS holo-ACP, catalyzed by His6-MCAT, was less efficient ( / was 10% of that of the S. coelicolor ACP). Incubation of MCAT with the serine specific agent PMSF caused inhibition of malonyl transfer to FAS ACPs, and an S97A MCAT mutant was incapable of catalyzing malonyl transfer. Our results show that in the reaction with FAS holo-ACPs the S. coelicolor MCAT is very similar to the E. coli MCAT paradigm in terms of its kinetic mechanism and active site residues. These results indicate that no other active site nucleophile is involved in catalysis as has been suggested to explain recently reported observations.