Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.
The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.
Liu
Zhi-Jie
ZJ
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. zjliu@ibp.ac.cn
Chen
Huizhong
H
Shaw
Neil
N
Hopper
Sherryll L
SL
Chen
Lirong
L
Chen
Siwei
S
Cerniglia
Carl E
CE
Wang
Bi-Cheng
BC
D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D013293:Enterococcus faecalis / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry*; D009601:Nitroreductases; D010802:Phylogeny
D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase