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, Takeshi Kawabata, Ken Nishikawa, Akio Tani, Kazuhide Kimbara, Fusako Kawai

    Research output: Contribution to journalArticlepeer-review

    15 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)4388-4396
    Number of pages9
    JournalApplied and environmental microbiology
    Volume72
    Issue number6
    DOIs
    Publication statusPublished - Jun 2006

    ASJC Scopus subject areas

    • Biotechnology
    • Food Science
    • Applied Microbiology and Biotechnology
    • Ecology

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