tailieunhanh - Báo cáo Y học: ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity

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. | Eur. J. Biochem. 269 2052-2059 2002 FEBS 2002 doi ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity Nadia J. Kershaw1 Heather J. McNaughton1 Kirsty S. Hewitson1 Helena Hernandez2 John Griffin3 Claire Hughes3 Philip Greaves3 Barry Barton3 Carol V. Robinson2 and Christopher J. Schofield1 1 Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory UK 2Oxford Centre for Molecular Sciences Central Chemistry Oxford UK 3GlaxoSmithKline Pharmaceuticals Worthing West Sussex UK 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 w 15 of total soluble protein by SDS PAGE analysis índícatíng it was lot 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 a2b2 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. .