title: A novel pyrroloquinoline quinone-dependent 2-keto-D-glucose dehydrogenase from Pseudomonas aureofaciens.
abstract: A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.
authors: Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
authors: Kiwamu
authors: K
authors: Umezawa
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors: Kouta
authors: K
authors: Takeda
authors: Department of Biomaterials Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors: Takuya
authors: T
authors: Ishida
authors: Department of Biomaterials Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors: Naoki
authors: N
authors: Sunagawa
authors: Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
authors: Akiko
authors: A
authors: Makabe
authors: Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors: Kazuo
authors: K
authors: Isobe
authors: Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
authors: Keisuke
authors: K
authors: Koba
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors: Hiroyuki
authors: H
authors: Ohno
authors: Department of Biomaterials Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors: Masahiro
authors: M
authors: Samejima
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors: Nobuhumi
authors: N
authors: Nakamura
authors: Department of Biomaterials Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors: Kiyohiko
authors: K
authors: Igarashi
authors: Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan ymakoto@cc.tuat.ac.jp.
authors: Makoto
authors: M
authors: Yoshida
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005948:Glucose Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D045542:PQQ Cofactor / Q000378:metabolism*; D010802:Phylogeny; D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D045542:PQQ Cofactor; D005948:Glucose Dehydrogenases
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