Product-substrate engineering by bacteria: Studies on clavaminate synthase, a trifunctional dioxygenase

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

“…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).…”

“…Coupled CAS2/ORF17 Activity Assay-The CAS2 assay was modified from that reported by Lloyd et al (24). The final incubation mixture contained 100 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol, 10 mM 2-OG, 2 mM FeSO 4 , 50 g of CAS2, and 2.5 mM synthetic racemic proclavaminic acid (24) in a final volume of 50 l. The assay mixture was incubated at 37°C in a water bath.…”

“…␤-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.…”

Mechanistic Studies on Three 2-Oxoglutarate-dependent Oxygenases of Flavonoid Biosynthesis

“…18 O labeling studies with prokaryotic 2OG oxygenases have demonstrated that during hydroxylation reactions less than stoichiometric incorporation of oxygen into the hydroxyl group can occur (59,60). This is thought to be due to solvent exchange of an iron-oxygen intermediate, mammalian type I prolyl-4-hydroxylase (62), and hypoxic inducible factor hydroxylases (63,64), show that Ͼ90% input of oxygen from dioxygen occurs on hydroxylation of their substrates.…”

Non‐Heme Fe(IV)—Oxo Intermediates

“…59 Clavaminate synthase (CAS) from Streptomyces clavuligerus performs three two-electron oxidation reactions in the biosynthesis of clavulanic acid: hydroxylation, oxidative ring closure and dehydrogenation (Scheme 8). [60][61][62][63][64][65] The hydroxylation reaction is believed to proceed via the canonical mechanism involving H-atom abstraction by the Fe(IV)-oxo intermediate, followed by hydroxyl radical rebound. It has been proposed that a different mode of reactivity of the Fe(III)-OH/substrate radical state yields the second and third reactions.…”