Site-directed mutagenesis of glycine 99 to alanine in L-lactate monooxygenase from Mycobacterium smegmatis.

L-Lactate monooxygenase (LMO) from Mycobacterium smegmatis was mutated at glycine 99 to alanine, and the properties of the resulting mutant (referred to as G99A) were studied. Mutant G99A of LMO was designed to test the postulate that the smaller glycine residue in the vicinity of the alpha-carbon methyl group of lactate in wild-type LMO has less steric hindrance, leading to the retention and oxidative decarboxylation of pyruvate in the active site, a unique property of LMO in contrast to other members of the FMN-dependent oxidase/dehydrogenase family. G99A has been shown to be readily reduced by L-lactate at a rate similar to that of the wild-type enzyme. The binding of pyruvate to reduced G99A is 4-fold weaker than that to the wild-type enzyme. A dramatic change of this mutation is that G99A has a much lower oxygen reactivity than the wild-type enzyme. Pyruvate-bound reduced G99A reacts with O2 at a rate approximately 10(5)-fold slower than the wild-type enzyme, and free reduced G99A reacts with O2 at a rate approximately 100-fold slower than the wild-type enzyme. Due to the very low oxygen reactivity of the pyruvate-bound reduced enzyme, G99A has been shown to catalyze the oxidation of L-lactate to pyruvate and hydrogen peroxide instead of acetate, carbon dioxide, and water, the normal decarboxylation products of pyruvate and hydrogen peroxide. Thus, the mutation alters the enzyme from its L-lactate monooxygenase activity to L-lactate oxidase activity. However, compared with L-lactate oxidase, G99A has a much lower reactivity toward oxygen. Our results also reveal that the small steric change around N-5 of the flavin causes a profound change in the electronic distribution in the catalytic cavity of the enzyme and imply that electrostatic interactions in the active site provide an important factor for control of O2 reactivity.