title: Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site.
abstract: 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 (His(6)) fusion protein in high yield. The His(6)-MCAT was purified to homogeneity using standard techniques, and kinetic analysis of the malonylation of S. coelicolorFAS holo-ACP, catalyzed by His(6)-MCAT, gave K(infinity) (M) values of 73 (ACP) and 60 microM (malonyl CoA). A catalytic constant k (infinity) (M) of 450 s(-1) and specificity constants k (infinity) (M)/K (infinity) (M) of 6.2 (ACP) and 7.5 microM(-1) s(-1) (malonyl CoA) were measured. Malonyl transfer to the E. coli FAS holo-ACP, catalyzed by His(6)-MCAT, was less efficient (k (infinity) (M)/K (infinity) (M) 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.
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, U.K.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Szafranska
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Hitchman
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Cox
authors.forename: John
authors.initials: J
authors.lastname: Crosby
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Simpson
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics; D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
chemical_list: D000213:Acyl Carrier Protein; D012694:Serine; D000217:Acyltransferases; C077402:3-ketoacyl-acyl carrier protein synthase III; D051080:Acyl-Carrier Protein S-Malonyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D064429:Fatty Acid Synthases