PHENYLGLYOXYLATE-NAD(-EVANSII() OXIDOREDUCTASE (COA BENZOYLATING), A NEW ENZYME OF ANAEROBIC PHENYLALANINE METABOLISM IN THE DENITRIFYING BACTERIUM AZOARCUS)

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic met abolism of phenylalanine and phenylacetate. It is formed by alpha-oxid ation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxyla ted by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central inter mediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizi ng enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxyla te : acceptor oxidoreductase was purified and studied. The oxygen-sens itive enzyme reduced both NAD(+) and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the nati ve mass as determined by gel filtration was 370 kDa, suggesting an alp ha(2) beta(2) gamma(2) delta(2) epsilon(2) composition. Phenylglyoxyla te : acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (K-m 45 mu M) and coenzyme A (K-m 55 mu M); 2-oxoisovalerate was oxidized with 15 % of the rate. Th e turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H : viologen dye tr anshydrogenation reaction, NAD(H) being the preferred coenzyme. It als o catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha(2) beta(2) gamma(2) delta(2), as reported for archa eal 2-oxoacid : ferredoxin oxidoreductases; this complex is able to re duce viologen dyes. The holoenzyme contains in addition an epsilon(2) unit that catalyzes the transfer of electrons from a small ferredoxin- like subunit of the core complex to NAD(+); this unit also catalyzes t he transhydrogenase reaction, carries FAD and resembles ferredoxin : N AD(P)(+)-oxidoreductase.