Abstract

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.

Highlights

  • From the Department Sciences, University Texas 77204-5500 of Biochemical and Biophysical of Houston, Houston, Flavin-dependent external monooxygenases and oxidases could catalyze the same flavin oxidation reaction involving distinct mechanisms

  • 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, highlighting the subtlety of enzyme active site structure in relation to catalysis and the versatility of enzyme evolution

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Summary

Both the hydroxylase and pseudooxidase pathways involve a

Equations 1 and 2b depict the same overall reaction, no FHOOH has ever been detected for any flavo-oxidases. A heterodimer designated a/3, catalyzes the overall reactions shown in pathways A and B in Scheme. In the sequence of major reaction steps, luciferase-bound FMNHz (I) reacts with oxygen to form the 4a-hydroperoxy FMN intermediate (II), which subsequently reacts with RCHO to emit bioluminescence following the light pathway (A). Intermediate II decays in the absence of aldehyde following a dark pathway (B) to generate FMN, Hz02 with very little or no light emission [8, 9]. V. harveyi luciferase CUC~S’~’ by methyl p-nitrobenzene sulfonate and have found that the modified enzyme retains the ability to bind aldehyde and FMNH2, but the formation of the peroxyflavin intermediate II is impaired [13]. Modified luciferase is active in catalyzing the oxidation of FMNHz or aldehyde in a dark reaction should be addressed. We have conducted site-directed mutagenesis to replace the CUC~S’~~with either an alanine (cuC106A) or a valine (cuC106V) to gain further insights into the luciferase structure-function relationship

PROCEDURES
SCHEME I
Mutagenesis of Luciferase
RESULTS AND DISCUSSION
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