Quinoline 2-oxidoreductase and 2-oxo-1,2-dihydroquinoline 5,6-dioxygenase from Comamonas testosteroni 63. The first two enzymes in quinoline and 3-methylquinoline degradation.
The enzymes catalysing the first two steps of quinoline and 3-methylquinoline degradation by Comamonas testosteroni 63 were investigated. Quinoline 2-oxidoreductase, which catalyses the hydroxylation of (3-methyl-)quinoline to (3-methyl-)2-oxo-1,2-dihydroquinoline, was purified to apparent homogeneity. The native enzyme, with a molecular mass of 360 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, iron, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8, the enzyme belongs to the molybdo-iron/sulfur flavoproteins. Molybdopterin cytosine dinucleotide is the organic part of the pterin molybdenum cofactor. Comparison of N-terminal amino acid sequences revealed similarities to a number of procaryotic molybdenum-containing hydroxylases. Especially the N-termini of the beta-subunits of the quinoline 2-oxidoreductases from Comamonas testosteroni 63, Pseudomonas putida 86, and Rhodococcus spec. B1, and of quinoline-4-carboxylic acid 2-oxidoreductase from Agrobacterium spec. 1B showed striking similarities. Further degradation of (3-methyl-)2-oxo-1,2-dihydroquinoline proceeds via dioxygenation at the benzene ring, i.e. at 5,6-position [Schach, S., Schwarz, G., Fetzner, S. & Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 175-181]. 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase was partially purified; 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 molecular 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-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is inhibited by sulfhydryl-blocking agents, by metal-chelating agents, and by the flavin analogues quinacrine and acriflavin.
Schach
S
S
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany.
Tshisuaka
B
B
Fetzner
S
S
Lingens
F
F
D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D004795:Enzyme Stability; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011804:Quinolines / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D011804:Quinolines; C081194:3-methylquinoline; C037219:quinoline; D010088:Oxidoreductases; D010105:Oxygenases; C095965:2-oxo-1,2-dihydroquinoline 5,6-dioxygenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C068667:quinoline 2-oxidoreductase