ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity

Abstract

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.

Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

“…Sequences TLLTFFA (amino acids 181 to 187) and TLLVVL (amino acids 179 to 184) were obtained from the Oat2 and ArgJ bands, respectively, purified from the gel. These results indicate that autoprocessing occurs in both cases at a threonine residue, which agrees with the hypothesis of Marc et al (15) and with the result described by Kershaw et al (11) for Oat2 while this work was in progress. Both proteins appear to belong to the N-terminal nucleophile hydrolase class of self-processing acetyltransferases (3).…”

“…Nothing is known about the ability of Oat1 to convert arginine to N-acetylarginine, and the additive action of both enzymes may have a more important channeling role. N-Acetylarginine has been reported to be a substrate of the clavaminate synthase (11). However, the kinetic mechanisms described for the ␤-lactam synthetase, the next enzyme in the pathway, do not support the use of N-acetylarginine as a substrate (2).…”

Crystal Structure of Carbapenem Synthase (CarC)

“…The level of substrate conversion effected by CarA with the trans-CMP was low compared with that of ␤-lactam synthetase (from the clavulanic acid biosynthesis pathway) with its natural substrate, possibly indicating an alternative in vivo substrate (see Scheme 1) or that a multiprotein complex is required to effect full activity (11). The organization of ␤-lactam biosynthesis proteins into a metabolon has also been suggested for clavulanic acid (40).…”

Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways

“…2 and Table 1) obtained by replacement with either the aph gene (conferring kanamycin resistance) or the apr gene (conferring apramycin resistance) were used for this study. They include mutants with deletions of the ceaS (pyc) gene, which encodes the carboxyethylarginine synthase (27); the bls gene, which encodes the ␤-lactam synthetase (2); the cla-ORF6 gene, which encodes a protein with ornithine acetyltransferase activity (7,11,28); the car gene, which encodes clavaldehyde reductase (26); the cyp gene, which encodes the cytochrome P450 involved in clavulanic acid formation (14,17); the two genes cla-ORF12 and cla-ORF15, which are located downstream of cyp (16,17); as well as mutants blocked in genes coding for the two ␤-lactam biosynthesis regulatory proteins, ccaR (25) and claR (26). In addition, the holomycin production of double mutants with disruptions in the ccaR and ORF6 genes was also studied.…”