The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at 15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to > 95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate a and b subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX-(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an a 2 b 2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the b subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the b subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major b subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate, but not the formation of N-acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N-acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the b subunit.

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-265β-Lactam Synthetase: Implications for β-Lactamase Evolution. β-Lactam synthetase I catalyzes the cyclization of (I) to (II) during clavulanic acid biosynthesis. -(MCNAUGHTON, H. J.; THIRKETTLE, J. E.; ZHANG, Z.; SCHOFIELD, C. J.; JENSEN, S. E.; BARTON, B.; GREAVES, P.; Chem.