Microbial metabolism of quinoline and related compounds. XVIII. Purification and some properties of the molybdenum- and iron-containing quinaldic acid 4-oxidoreductase from Serratia marcescens 2CC-1.
Serratia marcecens 2CC-1 utilizes quinaldic acid (quinoline 2-carboxylic acid) as sole source of carbon, nitrogen and energy. Growth of strain 2CC-1 on quinaldic acid as well as on nicotinic acid and hypoxanthine was inhibited completely by the molybdate antagonist tungstate, whereas growth on kynurenic acid and 6-hydroxynicotinic acid was not affected by tungstate. The synthesis of the molybdenum-containing hydroxylases quinaldic acid 4-oxidoreductase and nicotinic acid 6-oxidoreductase was found to be inducible. In addition, Serratia marcescens 2CC-1 produced a constitutively expressed xanthine oxidoreductase. Quinaldic acid 4-oxidoreductase was purified 1075-fold with a recovery of 5%. For catalytic activity, artificial electron acceptors were necessary. The 95-100-kDa enzyme was a heterodimer with subunit molecular masses of 75-80 kDa and 18-19 kDa. Quinaldic acid 4-oxidoreductase contained 2.3-3.7 g atom of iron and 0.5-0.6 g atom of molybdenum per mol of enzyme. The absorption spectrum exhibited maxima at 280 nm, 334 nm, 480 nm and a shoulder at 550 nm, with A280/A334 = 4.8, A280/A450 = 10.0, A280/A480 = 9.4, and A450/A550 = 1.6, suggesting the absence of a flavin cofactor. Acridine, quinacrine, ethylenediaminetetraacetate, 2,2'-dipyridyl, 1,10-phenanthroline and iodoacetate did not affect enzyme activity. p-Hydroxymercuribenzoate, m-arsenite, cyanide and methanol were effective inhibitors of quinaldic acid 4-oxidoreductase. Cyanide-inhibited enzyme was reactivated by treatment with S2-, indicating the presence of a pterin molybdenum cofactor with a monooxo-monosulfidotype molybdenum center. Quinaldic acid 4-oxidoreductase showed a very high substrate specificity, quinaldic acid being the only substrate found to be transformed significantly.
Fetzner
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Institut für Mikrobiologie der Universität Hohenheim, Stuttgart, Germany.
Lingens
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F
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000032:analysis*; D007526:Isoelectric Point; D007700:Kinetics; D007736:Kynurenic Acid / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis*; D011804:Quinolines / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology* / Q000254:growth & development; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014414:Tungsten / Q000494:pharmacology; D017973:Tungsten Compounds
D011804:Quinolines; D017973:Tungsten Compounds; D008982:Molybdenum; D007501:Iron; D006899:Mixed Function Oxygenases; C082851:quinaldic acid 4-oxidoreductase; D007736:Kynurenic Acid; C008898:quinaldic acid; C045951:tungstate; D014414:Tungsten