title: Catalytic mechanism of short ethoxy chain nonylphenol dehydrogenase belonging to a polyethylene glycol dehydrogenase group in the GMC oxidoreductase family.
abstract: Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor.
authors.affiliation: Urban and Environmental Science College, Liaoning Normal University, Dalian 116029, China. fkawai@kit.ac.jp.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ohta
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawabata
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D002794:Choline / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D012230:Rhizobiaceae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D042301:Sphingomonadaceae / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010636:Phenols; C025256:nonylphenol; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C027119:polyethylene glycol dehydrogenase; D005947:Glucose; D002794:Choline; D000432:Methanol