Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism

Abstract

Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P)H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P)H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of ∼3.5 Å to N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.

Azoreductase: a key player of xenobiotic metabolism

“…Previously, similar mechanism was proposed for reduction of FMN by NADH via hydride ion (Nissen et al 2008). Yu et al (2014) supported this existing mechanism with some more details. They determined the crystal structures of AzrA and AzrC complexed with Cibacron Blue (CB), Acid Red 88 (AR88), and Orange I (OI) but were unable to provide the binding of NAD(P)H.…”

“…B29 also exist as homodimers (Fig. 2) (Ito et al 2008;Yu et al 2014). Recently, azoreductases were investigated from alkaliphilic and neutrophilic bacterial strains having the monomeric appearance on size-exclusion column chromatography Yang et al (2013) and SDS PAGE (Misal et al 2011(Misal et al , 2013(Misal et al , 2014.…”

“…3). This known azo dye reduction mechanism always involves the reducing equivalents, e.g., FMN, NADH, and NADPH, which can reduce the azo compounds to a hydrazine in the first phase, and further the resulting hydrazines are converted into two amines (Nissen et al 2008;Wang et al 2010;Yang et al 2013;Yu et al 2014). However, an electron-transfer mechanism during the reduction process in flavin-free azoreductases is not well established by experimental methods.…”

A novel recombinant AzrC protein proposed by molecular docking and in silico analyses to improve azo dye's binding affinity

“…B29 are homodimeric proteins which contain two moles of FMN per mole of dimeric protein. AzrC contains a higher hydrophobicity and a smaller active-site pocket in comparison with AzrA (Yu et al, 2014). In the present study, a molecular docking analysis was performed to evaluate the detailed binding mode between AzrC with four different azo dyes and also to understand the specificity of substrate for AzrC.…”

“…AzrC protein is a FMN-containing azoreductase and also NADH-dependent oxidoreductases. It contains a homodimeric structure with two moles of FMN as a non-covalent prosthetic group per mole of dimeric protein (Roberts et al, 2011;Yu et al, 2014). In the present study, a molecular docking analysis to investigate the detailed binding mode between AzrC with four main azo dyes was performed.…”