ABSTRACT
The acyl carrier protein (ACP) of Escherichia coli was converted to acyl-ACP by imidazole-catalyzed S-acylation with N-acylimidazole. The acylation was specific to the sulfhydryl group; no acylation of tyrosine or amino groups of the protein occurred. The acyl-ACP substrates synthesized had a native structure as determined by gel electrophoresis, hydrophobic chromatography, and enzymatic activity. N-Acylimidazoles are readily synthesized and permit preparation of those acyl-ACP substrates that cannot be produced enzymatically.
Subject(s)
Acyl Carrier Protein/metabolism , Escherichia coli/enzymology , Esters/chemical synthesis , Kinetics , Structure-Activity Relationship , Substrate Specificity , Sulfhydryl CompoundsABSTRACT
Beta-Ketoacyl-acyl carrier protein synthases I and II of Escherichia coli were purified and characterized. Synthase I was shown to have a molecular weight of 80,000 +/- 5,000 and to be composed of two similarly sized subunits. Synthase II had a molecular weight of 85,000 +/- 5,000 and also was apparently homodimeric. Gel electrophoresis of partial proteolytic digests demonstrated that synthases I and II share few if any common peptides. Synthases I and II also were shown to be unrelated by immunological criteria. An improved assay for beta-ketoacyl-acyl carrier protein synthase activity gave kinetic parameters for synthases I and II at both 27 degrees C and 37 degrees C using five long chain acyl-acyl carrier protein substrates. The properties of synthase II are consistent with the proposed role of this enzyme in the modulation of fatty acid synthesis by temperature. fabF mutants of E. coli lack synthase II. The fabF locus was mapped at min 24.5 of the E. coli genetic map and the clockwise map order was found to be pyrC, fabD, fabF, purB.
Subject(s)
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/metabolism , Acyltransferases/metabolism , Escherichia coli/enzymology , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics , Escherichia coli/genetics , Genotype , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Molecular Weight , Species SpecificityABSTRACT
Cvc- mutants of Escherichia coli are deficient in the synthesis of cis-vaccenic acid and in the temperature control of fatty acid synthesis. In this communication, it is demonstrated that these mutants lack beta-ketoacyl-acyl carrier protein synthase II. The deficiencies in cis-vaccenate synthesis and synthase II are shown to be due to a lesion in the same gene, fabF. Lesions in the fabF gene are found to affect growth only when the strain also carries a lesion in the fabB gene, the structural gene for beta-ketoacyl-acyl carrier protein synthase I.