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

1 Faculty of Nutrition, Kobe Gakuin University, Kobe, Hyogo, 651-2180, Japan 2 Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan 3 Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan Correspondence Fusako Kawai fkawai{at}rib.okayama-u.ac.jp 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. Abbreviations: OPH, oxidized PVA hydrolase; PPG, polypropylene glycol; PQQ, pyrroloquinoline quinone; PVA, poly(vinyl alcohol); PVADH, poly(vinyl alcohol) dehydrogenase; QH-ADH, quinohaemoprotein alcohol dehydrogenase; QH-PVADH, quinohaemoprotein poly(vinyl alcohol) dehydrogenase Sequence alignments of type II QH-PVADHs with type II QH-ADHs are available as supplementary data with the online version of this paper.