Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis

ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

“…Attempts to couple BLS, PAH, and CAS2 using N 2 -(2-carboxyethyl)arginine as the initial substrate were similarly unsuccessful. In this instance, the major product observed was deoxyproclavaminic acid, produced by the hydrolysis of the guanidino group of deoxyguanidinoproclavaminic acid as catalyzed by PAH (21,28). This is consistent with the observation that deoxyproclavaminic acid has been reported to be a relatively poor substrate for CAS2 (24).…”

“…Coupled Assay Procedures-Recombinant CEAS, BLS, PAH, and CAS2 were prepared as reported (14,15,21,24,48). A typical coupled enzyme incubation mixture contained 150 mM Tris-HCl (pH 8.0), 12 M CEAS, BLS, PAH, and CAS2 with appropriate cofactors (30 mM D/L-glyceraldehyde-3-phosphate, 1.5 mM thiamin diphosphate, 10 mM MgCl 2 , 5 mM ATP, 0.5 mM MnCl 2 , 10 mM FeSO 4 , and 10 mM 2-OG) and substrates (30 mM L-arginine or 10 mM deoxyguanidinoproclavaminic acid), in a final volume of 100 l. The assay mixtures were incubated at (DDL_HAEIN, P44405); carbamoyl phosphate synthetase subfamily highlighted in green, E. coli (1A9X (51), P00968), Saccharomyces cerevisiae (CARB_YEAST, P03965), and Trichosporon cutaneum (CARB_TRICU, P46056).…”

“…␤-Lactam formation is catalyzed by ␤-lactam synthetase (BLS) (15,16), to give deoxyguanidinoproclavaminic acid (17), which is then converted in four steps to (3S,5S)-clavaminic acid. Three of these steps are catalyzed by a single 2-oxoglutarate (2-OG)-dependent oxygenase, clavaminic acid synthase (CAS2) (10, 18 -20), and one is catalyzed by proclavaminate amidinohydrolase (PAH) (21), to give the bicyclic clavam ring system (22)(23)(24). Crystal structures have been reported for the first four enzymes in the pathway (25)(26)(27)(28) as well as for the gene product of orf6 (OAT2), an ornithine acetyltransferase (29,30) proposed to be involved in the biosynthesis of the L-arginine feedstock for the pathway.…”

β-Lactam synthetase: A new biosynthetic enzyme

“…Compared with controls, a new peak was observed, which increased in a time-dependent manner and coeluted with a standard sample of DGPC. H-NMR analysis of this new product showed it to correspond to DGPC (4) (27).…”

Two Oligopeptide-Permease-Encoding Genes in the Clavulanic Acid Cluster of Streptomyces clavuligerus Are Essential for Production of the β-Lactamase Inhibitor

“…Genes for clavulanic acid biosynthesis are located adjacent to those of the cephamycin C cluster in S. clavuligerus (7,8,33), forming the so-called ␤-lactam supercluster. Several enzymes encoded by these genes have been purified to homogeneity, yielding information on different steps of the pathway (2,9,29,34), and the ␤-lactam synthetase and clavaminate synthetase have been crystallized (17,35). However, the studies on clavulanic acid biosynthesis are confused by the presence of two sets of paralogous genes that encode the early enzymes of the pathway (8).…”