MICROBIAL-METABOLISM OF QUINOLINE AND RELATED-COMPOUNDS .20. QUINALDIC ACID 4-OXIDOREDUCTASE FROM PSEUDOMONAS SP AK-2 COMPARED TO OTHER PROKARYOTIC MOLYBDENUM-CONTAINING HYDROXYLASES

Quinaldic acid 4-oxidoreductase from Pseudomonas sp. AK-2 catalyses th e hydroxylation of quinoline 2-carboxylic acid (quinaldic acid) to 4-h ydroxyquinoline 2-carboxylic acid (kynurenic acid) with concomitant re duction of a suitable electron acceptor. An analogous hydroxylation in para-position relative to the N-heteroatom was only recently describe d for quinaldine 4-oxidoreductase (de Beyer & Lingens, 1993, Biol. Che m. Hoppe-Seyler 374, 101-110) and for quinaldic acid 4-oxidoreductase from Serratia marcescens 2CC-1 (Fetzner & Lingens, 1993, Biol. Chem. H oppe-Seyler 374, 363 - 376). Quinaldic acid 4-oxidoreductase from Pseu domonas putida AK-2 was purified 78-fold to electrophoretic homogeneit y with a recovery of 22%. The native enzyme (300 kDa) was composed of three subunits with molecular masses of 90, 34 and 20 kDa, indicating an alpha2beta2gamma2 structure. Quinaldic acid 4-oxidoreductase contai ned FAD, molybdenum, iron and acid-labile sulfur in a ratio of 2:2:8:8 . Molybdenum is probably associated with molybdopterin cytosine dinucl eotide as organic part of the pterin molybdenum cofactor. The absorpti on spectrum of quinaldic acid 4-oxidoreductase exhibited the typical f eatures of a molybdo-iron/sulfur-flavoprotein, namely, maxima at 274 n m, 340 nm and 450 nm, a shoulder at 550 nm, a ratio A280/A450 of 4.7 a nd a ratio A450/A550 of 3.5. The enzyme was susceptible to inactivatio n by methanol, sodium m-arsenite, p-hydroxymercuribenzoate, and potass ium cyanide. Cyanide caused an alteration at 320 nm in the absorption spectrum, typical for the change in the coordination sphere of the mol ybdenum. Enzyme inactivated with cyanide was reactivated to 74% by inc ubation with sulfide. Thus, quinaldic acid 4-oxidoreductase possesses a monooxo-monosulfido-type molybdenum center.