Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase

Rie Hirota-Mamoto, Ryoko Nagai, Shinjiro Tachibana, Masaaki Yasuda, Akiwo Tani, Kazuhide Kimbara, Fusako Kawai

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54-25% identity), and the quinohaemoprotein alcohol dehydrogenases (QH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25-29% identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SD and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SD are the reverse of those of QH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.

Original languageEnglish
Pages (from-to)1941-1949
Number of pages9
JournalMicrobiology
Volume152
Issue number7
DOIs
Publication statusPublished - Jul 2006

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Sphingomonas
Alcohol Dehydrogenase
Organism Cloning
Oxidoreductases
Gene Expression
Amino Acid Sequence
His-His-His-His-His-His
Heme
Azoarcus
Comamonas testosteroni
Cupriavidus necator
Xanthomonas
Genes
Pseudomonas putida
Glycols
Nucleic Acid Databases
Hydrolases
Enzymes
Operon
Pseudomonas

ASJC Scopus subject areas

  • Microbiology

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Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase. / Hirota-Mamoto, Rie; Nagai, Ryoko; Tachibana, Shinjiro; Yasuda, Masaaki; Tani, Akiwo; Kimbara, Kazuhide; Kawai, Fusako.

In: Microbiology, Vol. 152, No. 7, 07.2006, p. 1941-1949.

Research output: Contribution to journalArticle

Hirota-Mamoto, Rie ; Nagai, Ryoko ; Tachibana, Shinjiro ; Yasuda, Masaaki ; Tani, Akiwo ; Kimbara, Kazuhide ; Kawai, Fusako. / Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase. In: Microbiology. 2006 ; Vol. 152, No. 7. pp. 1941-1949.
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abstract = "A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54-25{\%} identity), and the quinohaemoprotein alcohol dehydrogenases (QH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25-29{\%} identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SD and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SD are the reverse of those of QH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.",
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T1 - Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase

AU - Hirota-Mamoto, Rie

AU - Nagai, Ryoko

AU - Tachibana, Shinjiro

AU - Yasuda, Masaaki

AU - Tani, Akiwo

AU - Kimbara, Kazuhide

AU - Kawai, Fusako

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