QUINOLINE 2-OXIDOREDUCTASE AND 2-OXO-1,2-DIHYDROQUINOLINE 5,6-DIOXYGENASE FROM COMAMONAS-TESTOSTERONI-63 - THE FIRST 2 ENZYMES IN QUINOLINEAND 3-METHYLQUINOLINE DEGRADATION

The enzymes catalysing the first two steps of quinoline and 3-methylqu inoline degradation by Comamonas testosteroni 63 were investigated. Qu inoline 2-oxidoreductase, which catalyses the hydroxylation of (3-meth yl-)quinoline to (3-methyl-)2-oxo-1,2-dihydroquinoline, was purified t o apparent homogeneity. The native enzyme, with a molecular mass of 36 0 kDa, is composed of three non-identical subunits (87, 32, and 22 kDa ), occurring in a ratio of 1.16:1:0.83. Containing FAD, molybdenum, ir on, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8, the enzyme belongs to the molybdo-iron/sulfur flavoproteins. Molybdopteri n cytosine dinucleotide is the organic part of the pterin molybdenum c ofactor. Comparison of N-terminal amino acid sequences revealed simila rities to a number of procaryotic molybdenum-containing hydroxylases. Especially the N-termini of the beta-subunits of the quinoline 2-oxido reductases from Comamonas testosteroni 63, Pseudomonas putida 86, and Rhodococcus spec. B1, and of quinoline-4-carboxylic acid 2-oxidoreduct ase from Agrobacterium spec. 1B showed striking similarities. Further degradation of (3-methyl-)2-oxo-1,2-dihydroquinoline proceeds via diox ygenation st the benzene ring, i.e. at 5,6-position [Schach, S., Schwa rz, G., Fetzner, S. & Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 175-181]. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase was partially pu rified; NADH and oxygen are required for the reaction, and the enzymic activity is enhanced 1.5-fold by addition of Fe2+ ions. Unexpectedly, this aromatic ring dioxygenase did not separate into distinct protein components, but is apparently a single-component enzyme. The molecula r mass was estimated to be about 260 kDa. 2-Oxo-1,2-dihydroquinoline 5 ,6-dioxygenase is very thermolabile. However, dithioerythritol and low concentrations of substrate had a moderately stabilizing effect. 2-Ox o-1,2-dihydroquinoline 5,6-dioxygenase is inhibited by sulfhydryl-bloc king agents, by metal-chelating agents, and by the flavin analogues qu inacrine and acriflavin.