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

Abstract

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 Q 10 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.