Purification, characterization, DNA sequence and cloning of a pimeloyl-CoAsynthetase from Pseudomonas mendocina 35

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and ch aracterized, the DNA sequence determined, and the gene cloned into Escheric hia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximate to 8.0, K-m values of 0.49 mM for pimelic acid, 0.18 mM for Co A and 0.72 mM for ATP, a subunit M-r of approximate to 80 000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatograp hy. The specific activity of the purified enzyme was 77.3 units/mg of prote in. The enzyme was not absolutely specific for pimelic acid. The relative a ctivity for adipic acid (C-6) was 72% and for azaleic acid (C-9) was 18% of that for pimelic acid (C-7). The N-terminal amino acid was blocked to amin o acid sequencing, but controlled proteolysis resulted in three peptide fra gments for which amino acid sequences were obtained. An oligonucleotide gen e probe corresponding to one of the amino acid sequences was synthesized an d used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pime loyl-CoA synthetase. The pauA gene, which codes for a protein with a theore tical M-r of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fu lgidus, Methanococcus: jannaschii, Pyrococcus horikoshii, E. coli and Strep tomyces coelicolor, and some limited similarity to microbial succinyl-CoA s ynthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an ac tive enzyme.