Kinetic investigations on a flavoprotein oxygenase, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

In the presence of NADH and O2, 2-methyl-3-hydroxypyridine-5-carboxylate oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 catalyzes reductive oxygenation of 2-methyl-3-hydroxypyridine-5-carboxylate (Cpd I) to yield alpha-N-(acetylaminomethylene)succinic acid (Cpd A). Steady state kinetic data and studies with alternate substrates are consistent only with an ordered mechanism in which Cpd I binds first, followed by NADH; the first product, NAD+, is then released. This event is followed by oxygen binding, and finally release of the oxygenated and reduced cleavage product, Cpd A. This kinetic mechanism was confirmed by studying inhibition by NAD+, which binds competitively with oxygen, but not with NADH. The kinetic mechanism of this reaction resembles that proposed for bacterial flavin monooxygenases that catalyze hydroxylation of aromatic homocyclic compounds.