The gene pvaB encodes oxidized polyvinyl alcohol hydrolase of Pseudomonas sp. strain VM15C and forms an operon with the polyvinyl alcohol dehydrogenase gene pvaA

Department of Biotechnology, Tottori University, Tottori, Tottori 680-8552, Japan 1 Marine Biotechnology Institute, Kamaishi Laboratory, Kamaishi, Iwate 026-0001, Japan 2 Author for correspondence: Masayuki Shimao. Tel: +81 857 31 5268. Fax: +81 857 31 0881. e-mail: shimao{at}lemon.bio.tottori-u.ac.jp A 5·7 kbp Sph I fragment containing the polyvinyl alcohol (PVA) dehydrogenase gene pvaA and its 1·9 kbp 5'-flanking region was cloned from the PVA-degrading bacterium Pseudomonas sp. VM15C. The pvaB gene, encoding oxidized PVA hydrolase, was found in the region upstream of pvaA . Sequence data and expression studies indicated that pvaA and B constitute an operon in the order pvaBA . The pvaB gene encoded a protein of 379 amino acid residues (40610 Da), and a lipoprotein signal sequence and the lipase consensus sequence, Gly-X-Ser-X-Gly, characteristic of the active-site serine region in serine hydrolases, were detected in the deduced amino acid sequence. The pvaB product with the pvaA product constituted an enzyme system for the cleavage of PVA molecules. The pvaA product introduced ß-diketone groups into the PVA molecule, and the pvaB product hydrolysed these ß-diketone groups in oxidized PVA. The pvaB product also hydrolysed 4,6-nonanedione at a low rate, but not acetylacetone or 5-nonanone. It was completely inhibited by PMSF and was concluded to be a serine hydrolase. There were no proteins showing high similarity to the pvaB product in the databases, but minor similarity to a number of serine hydrolases including polyhydroxyalkanoate depolymerases was apparent. Keywords: oxidized polyvinyl alcohol hydrolase, PQQ-dependent polyvinyl alcohol dehydrogenase, pva operon, Pseudomonas sp. VM15C Abbreviations: DIG, digoxigenin-3- O -succinyl- -aminocaproyl-[5-(3-aminoallyl)-2'-deoxy-uridine-5'-triphosphate]; KPB, potassium phosphate buffer; OPH, oxidized polyvinyl alcohol hydrolase; PES, phenazine ethosulfate; PVA, polyvinyl alcohol; PVADH, polyvinyl alcohol dehydrogenase; PQQ, pyrroloquinoline quinone The DDBJ accession number for the sequence reported in this paper is AB008494.