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
Summary
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
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