Xenoestrogenic short ethoxy chain nonylphenol is oxidized by a flavoprotein alcohol dehydrogenase from Ensifer sp. strain AS08.
The ethoxy chains of short ethoxy chain nonylphenol (NPEO(av2.0), containing average 2.0 ethoxy units) were dehydrogenated by cell-free extracts from Ensifer sp. strain AS08 grown on a basal medium supplemented with NPEO(av2.0). The reaction was coupled with the reduction in 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and phenazine methosulfate. The enzyme (NPEO(av2.0) dehydrogenase; NPEO-DH) was purified to homogeneity with a yield of 20% and a 56-fold increase in specific activity. The molecular mass of the native enzyme was 120 kDa, consisting of two identical monomer units (60 kDa). The gene encoding NPEO-DH was cloned, which consisted of 1,659 bp, corresponding to a protein of 553 amino acid residues. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified NPEO-DH. The presence of a flavin adenine dinucleotide (FAD)-binding motif and glucose-methanol-choline (GMC) oxidoreductase signature motifs strongly suggested that the enzyme belongs to the GMC oxidoreductase family. The protein exhibited homology (40-45% identity) with several polyethylene glycol dehydrogenases (PEG-DHs) of this family, but the identity was lower than those (approximately 58%) among known PEG-DHs. The substrate-binding domain was more hydrophobic compared with those of glucose oxidase and PEG-DHs. The recombinant protein had the same molecular mass as the purified NPEO-DH and dehydrogenated PEG400-2000, NPEO(av2.0) and its components, and NPEOav10, but only slight or no activity was found using diethylene glycol, triethylene glycol, and PEG200.
Liu
Xin
X
Laboratory of Applied Microbiology, Research Institute for Bioresources, Okayama University, Kurashiki, 2-20-1 Chuo, Kurashiki, 710-0046, Japan. liuxin@server.rib.okayama-u.ac.jp
Tani
Akio
A
Kimbara
Kazuhide
K
Kawai
Fusako
F
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010636:Phenols / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012230:Rhizobiaceae / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D005420:Flavoproteins; D010636:Phenols; D011994:Recombinant Proteins; C025256:nonylphenol; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C027119:polyethylene glycol dehydrogenase