Three-dimensional Structure of AzoR from Escherichia coli

Abstract

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 Å resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.

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

“…AzoA shares features with the catalytic apparatus of other azoreductases, AzoR (Ito et al, 2006) from E. coli and paAzoR1 from Pseudomonas aeruginosa. FMN is bound to approximately the same regions of these proteins, making the overall conformation of FMN and the orientation of isoalloxazine moiety very similar in each; the si face of the isoalloxazine ring of FMN is solvent-accessible, whereas its re face is buried in the protein.…”

“…In the active site, the FMN sits inside a positively charged cavity formed by several stretches of residues; the si face of the isoalloxazine ring of FMN is solvent-accessible, whereas its re face is buried in the protein. The structural environment of the active site could be correlated with the mechanical view adopted previously for azoreductase (Ito et al, 2006;Liu et al, 2007;Wang et al, 2007).…”

“…Despite the large body of biochemical and 3D structural information available on bacterial FMN-dependent azoreductases (Chen et al, 2004(Chen et al, , 2005Ito et al, 2006;Liu et al, 2007;Wang et al, 2007), little is known about the role of specific amino acid residues involved in the flavin binding and catalytic mechanism. In this study, a computational protein-FMN interaction analysis was conducted, which identified Trp-105 on loop ,9 as a potential determinant for FMN stabilization.…”

“…Biochemical and 3D structural information is available for FMN-dependent azoreductases of Escherichia coli, Ent. faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Saccharomyces cereviseae (Chen et al, 2004(Chen et al, , 2005Ito et al, 2006;Liu et al, 2007;Wang et al, 2007).…”

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…Despite some of them sharing only minimal sequence identity, they have a common three dimensional fold (Fig. 1B) (Li et al, 1995;Ito et al, 2006;Wang et al, 2007;Ye et al, 2007;Binter et al, 2009).…”