An azoreductase, aerobic NADH-dependent flavoprotein discovered from Bacillus sp.: functional expression and enzymatic characterization.
The gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60-80 degrees C. The enzyme cleaved the model azo compound of methyl red [MR, 4'-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N'-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.
Ooi
Toshihiko
T
Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, 060-8628, Sapporo, Japan. ooi@dove-mc.eng.hokudai.ac.jp
Shibata
Takeshi
T
Sato
Reiko
R
Ohno
Hiroaki
H
Kinoshita
Shinichi
S
Thuoc
Tran Linh
TL
Taguchi
Seiichi
S
D000332:Aerobiosis; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005420:Flavoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009601:Nitroreductases; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D005420:Flavoproteins; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase