title: [Dehydrogenation by short ethoxy chain nonylphenol dehydrogenase from Ensifer sp. AS08].
abstract: OBJECTIVE
  To study dehydrogenation by short ethoxy chain nonylphenols dehydrogenase (sNPEO-DH) from Ensifer sp. AS08.
  
  
  METHODS
  We screened four amino acid residues of sNPEO-DH that are adjacent to the isoalloxazine ring of the coenzyme flavin adenine dinucleotide (FAD) by using multiple sequence alignment and homology modeling. Mutations were introduced by site-directed mutagenesis. The recombinant proteins were expressed, purified and assayed.
  
  
  RESULTS
  The relative activities of mutants N90A and N509A against the hydrophilic substrate PEG1000 decreased to 51% and 89%, respectively, and against the hydrophobic substrate NPEO(av10) decreased to 26% and 40%, respectively; indicating that N90 and N509 might be related to substrate binding. The relative activity of mutant H465A and N507A lost 90% and 100%, respectively; "stop-flow" experiments revealed that the processes of proton transfer from substrate to FAD and from FAD to enzyme were blocked in mutant N507A and H465A, respectively.
  
  
  CONCLUSIONS
  Amino acid residues N507 and H465 located at the activity center of sNPEO-DH and play roles as catalytic sites for the oxidative dehydrogenation of the substrates and FADH2, respectively.
authors.affiliation: College of Urban and Environmental Science, Liaoning Normal University, Dalian 116029, China. zhangruiyuan5337@163.com
authors.forename: Ruiyuan
authors.initials: R
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Guangfeng
authors.initials: G
authors.lastname: Zhou
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.forename: Guanghao
authors.initials: G
authors.lastname: Qiao
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D005420:Flavoproteins / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012230:Rhizobiaceae / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D010636:Phenols; D011994:Recombinant Proteins; C025256:nonylphenol; D010088:Oxidoreductases