Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis

Abstract

Enterococcus faecalis azoreductase (AzoA) is a very active enzyme with a broad spectrum of substrate specificity and is capable of degrading various azo dyes. The enzyme has an absolute requirement for reduced FMN, which delivers a total of four electrons from NADH to the substrate, resulting in the cleavage of the nitrogen double bond. In this study, we report the identification of amino acid residues critical for FMN binding in AzoA. FMN is stabilized by 22 amino acid residues, eight of which, Trp-105, Asn-106, Leu-107, Gly-150, Gly-151, Tyr-153, Asn-121 and Tyr-129, are involved in binding the FMN isoalloxazine ring. In silico analysis of the amino acid residues revealed that the Trp residue at position 105 of AzoA is the most likely significant contributor to the binding of FMN to the enzyme and is involved in FMN stabilization and destabilization. Site-directed mutagenesis analysis of Trp-105 was performed to determine the role of this amino acid residue in FMN binding and azo dye reductive activity. The mutant proteins were overexpressed in Escherichia coli and purified by anion-exchange and size-exclusion chromatography. The replacement of Trp-105 by the small side-chain amino acids Ala and Gly caused complete loss of both affinity for FMN and enzyme activity. Substitution of Tyr for Trp-105 did not significantly decrease the V max of the enzyme (22 % reduction). Substitutions with three bulky side-chain amino acids, Gln, Phe and His, produced enzymes with lower V max values (decreases of 68.2, 30.6 and 8.2-fold, respectively). However, these mutated enzymes maintained K m values similar to the wild-type enzyme. This study provides an insight into the catalytic properties of AzoA in FMN stabilization and enzyme activity.

Characteristics of majorEscherichia colireductases involved in aerobic nitro and azo reduction

“…In previous studies, MR reduction was followed by a kinetic decrease in absorbance at 430 nm (Chung et al 1992;Chen et al 2008;Macwana et al 2010;Feng et al 2012) ( Fig. 2a).…”

A Novel Mechanism for Azoreduction

“…47 The side chain of Tyr98, which forms stacking interactions with FMN and a water-mediated hydrogen bond to the FMN phosphate group, plays a vital role in FMN binding, as replacing it with glycine results in a loss of FMN binding. 48,49 In flavodoxins, a separate loop at the entrance of the active site is also involved in FMN binding. This loop comprises two glycine residues (Gly146 and Gly147; Fig.…”

Effects of Orange II and Sudan III azo dyes and their metabolites on Staphylococcus aureus

“…The initial step of degradation of azo dyes is reduction of the azo bond catalyzed by azoreductase. Azoreductases are commonly found in bacteria and have been purified and characterized from Bacillus subtilis [39], Escherichia coli [25, 26], Enterococcus faecalis [11, 34, 35], Pseudomonas aeruginosa [45, 47, 48], Saccharomyces cereviaeae [32], and Staphylococcus aureus [9]. The enzymes can be classified into three groups, flavin-dependent NADH-preferred azoreductase, flavin-dependent NADPH-preferred azoreductase, and flavin-free NADPH-preferred azoreductase [8].…”