Elicitation of an oxidase activity in bacterial luciferase by site-directed mutation of a noncatalytic residue.
Flavin-dependent external monooxygenases and oxidases could catalyze the same flavin oxidation reaction involving distinct mechanisms. To gain insights into enzyme structure-function relationship, site-directed mutagenesis was carried out for Vibrio harveyi luciferase, a monooxygenase. The substitution of the alpha subunit cysteine 106 by alanine shows unambiguously that the alphaCys106 is not essential to catalysis. The corresponding substitution by valine resulted in a substantial reduction of the bioluminescence activity correlatable with the induction of a new flavin oxidation activity typical for oxidases. These findings indicate that mutation of a single noncatalytic residue at the active center of a flavoenzyme could transform one enzyme type to another, thus highlighting the subtlety of enzyme active site structure in relation to catalysis and the versatility of enzyme evolution.
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Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5500.
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Herndon
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D002384:Catalysis; D003001:Cloning, Molecular; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014733:Vibrio / Q000201:enzymology*
D005486:Flavin Mononucleotide; D010088:Oxidoreductases; D008156:Luciferases