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Applied and Environmental Microbiology, June 2006, p. 4388-4396, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02174-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from Sphingopyxis terrae, Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants

Takeshi Ohta,1,{dagger} Takeshi Kawabata,2,{ddagger} Ken Nishikawa,2 Akio Tani,1 Kazuhide Kimbara,1 and Fusako Kawai1*

Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan,1 Center for Information Biology, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan2

Received 14 September 2005/ Accepted 17 March 2006

Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q10 was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.

* Corresponding author. Mailing address: Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan. Phone and fax: 81-86-434-1225. E-mail: fkawai{at}rib.okayama-u.ac.jp.

{dagger} Present address: Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.

{ddagger} Present address: Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

Applied and Environmental Microbiology, June 2006, p. 4388-4396, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02174-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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