Structure-based mutagenesis of the malonyl-CoA:acyl carrier protein transacylase from Streptomyces coelicolor.
Malonyl-CoA:acyl carrier protein transacylase (MAT) provides acyl-ACP thioesters for the biosynthesis of aromatic polyketides, and thus is the primary gatekeeper of substrate specificity in type II PKS. A recent report described the X-ray crystal structure of the Streptomyces coelicolor MAT and suggested active site residues which may be important for substrate selectivity [Keatinge-Clay, A. T., et al. (2003) Structure 11, 147-154]. Mutants were made to test the proposed roles of these residues, and the enzymes were characterized kinetically with respect to native and non-native substrates. The activity of the MAT was observed to be greatly attenuated in many of the observed mutants; however, the K(m) for malonyl-CoA was only modestly affected. Our results suggest the MAT uses an active site that is rigorously ordered around the acyl-thioester moiety of the acyl-CoA to facilitate rapid and efficient transacylation to an ACP. Our results also suggest that the MAT does not discriminate against alpha-substituted acyl-CoA thioesters solely on the basis of substrate size.
Koppisch
Andrew T
AT
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Khosla
Chaitan
C
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009285:Naphthoquinones / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D000095622:Benzoisochromanequinones
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D009285:Naphthoquinones; C018066:frenolicin B; D000095622:Benzoisochromanequinones; C494968:fabD protein, E coli