Conversion of L-lactate oxidase to a long chain alpha-hydroxyacid oxidase by site-directed mutagenesis of alanine 95 to glycine.
A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long chain alpha-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-alpha-hydroxyacids, alpha-hydroxy-n-butyric acid, alpha-hydroxy-n-valeric acid, etc., and also the aromatic alpha-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pKa of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.
Institute for Enzyme Research, University of Tokushima, Tokushima 770, Japan. vince_massey@mailgw.surg.med.umich.edu
K
K
Yorita
K
K
Aki
T
T
Ohkuma-Soyejima
T
T
Kokubo
H
H
Misaki
V
V
Massey
D000409:Alanine; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D004795:Enzyme Stability; D005998:Glycine; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D019289:Pyruvic Acid; D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; C021380:L-2-hydroxyacid oxidase; C021497:lactate 2-monooxygenase; D000409:Alanine; D005998:Glycine