Azo dyes, synthetic organic colorants, are extensively used in printing, in food, for clinical purposes and in the cosmetics industry because of their chemical stability and ease of synthesis and utility (Meyer, 1981
). Most azo dyes are released into the environment in waste water. They are not completely degraded even after waste-water treatment based on chemical procedures, which are expensive methods and often yield hazardous byproducts. In addition, the toxic and mutagenic properties of some azo dyes (Holme, 1984
) has led to a demand for efficient technology for their degradation. Biological degradation using microorganisms is a useful method for decomposing the dyes under mild conditions without the problems mentioned above (Robinson et al.
; Stolz, 2001
). Therefore, a detailed understanding of the mechanisms of dye-degrading enzymes will facilitate the development of biodegradation systems.
AzoR, an enzyme catalyzing the reductive cleavage of azo groups (—N=N—), was purified from Escherichia coli
(Nakanishi et al.
) in order to determine the molecular basis of the biodegradation method. Biochemical studies revealed that AzoR exists as a homodimer composed of 23 kDa (200 amino-acid) subunits, as shown by gel filtration. AzoR utilizes NADH but not NADPH as an electron donor and binds FMN as a flavin cofactor. The reaction follows a ping-pong mechanism requiring 2 mol NADH to reduce 1 mol methyl red (4′-dimethylaminoazobenzene-2-carboxylic acid), a typical azo dye, to 2-aminobenzoic acid and N,N
-phenylenediamine. However, details of the molecular mechanism of catalysis remain unknown.
Many types of bacterial cytoplasmic azoreductases have been isolated and characterized for the purpose of environmental biotechnology (Ghosh et al.
; Maier et al.
; Moutaouakkil et al.
; Rafii & Cerniglia, 1993
; Ramalho et al.
; Suzuki et al.
; Zimmermann et al.
). However, AzoR is different from other azoreductases reported thus far with respect to its substrate specificity, requirement for a flavin cofactor and type of electron donor. For example, the azoreductase purified from Bacillus
sp. by Suzuki and coworkers degrades azo compounds utilizing NADPH, but NADPH is ineffective as an electron donor for AzoR. The azoreductase purified from Pseudomonas
KF46 by Zimmermann and coworkers utilizes FAD as a flavin cofactor; however, AzoR utilizes FMN as a flavin cofactor. Although the physiological function of AzoR remains unknown, its importance has been deduced from the wide distribution of AzoR orthologues in bacteria, as has been revealed by genome projects, such as those on Bacillus subtilis
(Kunst et al.
), Streptomyces coelicolor
(Bentley et al.
), Salmonella typhimurium
(McClelland et al.
), Haemophilus influenzae
(Fleischmann et al.
) and Pseudomonas aeruginosa
(Stover et al.
). On the other hand, AzoR shows a moderate sequence homology in the active site to NQO1, originally called DT-diaphorase [NAD(P)H:quinone reductase; EC 18.104.22.168], a mammalian FAD-containing protein that also catalyzes the reduction of azo compounds (Bayney et al.
). NQO1 plays an important role in detoxification and has a protective effect against mutagenicity, carcinogenicity and other toxicities by utilizing its reduction activity (Benson et al.
; Chesis et al.
; Smith, 1999
). These facts imply that AzoR may play an important role in detoxification in bacteria.
AzoR is representative of a poorly characterized family of azo-dye reductases. Structure determination of AzoR is a first step toward the elucidation of their molecular mechanism of function. Here, we report the crystallization and preliminary X-ray crystallographic analysis and the search for heavy-atom derivatives for use in phasing. This is the first report of the crystallization of an FMN-dependent NADH-azo reductase.