Hirota-Mamoto Rie, Nagai Ryoko, Tachibana Shinjiro, Yasuda Masaaki, Tani Akio, Kimbara Kazuhide, Kawai Fusako
Faculty of Nutrition, Kobe Gakuin University, Kobe, Hyogo, 651-2180, Japan.
Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan.
Microbiology (Reading). 2006 Jul;152(Pt 7):1941-1949. doi: 10.1099/mic.0.28848-0.
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.
基于从鞘氨醇单胞菌属菌株113P3纯化的周质聚（乙烯醇）（PVA）脱氢酶（PVADH）的N端氨基酸序列以及PVADH基因（pvaA，GenBank登录号AB190288）的序列，克隆了一个基因。用六组氨酸标记的重组PVADH在大肠杆菌中表达并纯化至均一。重组酶具有与鞘氨醇单胞菌属菌株113P纯化酶相同的特性。除了PVA外，重组PVADH还可以氧化二醇，如聚丙二醇、1,3 - 丁烷/环己二醇和2,4 - 戊二醇，但不能氧化伯醇或仲醇。重组PVADH的氨基酸序列与假单胞菌属菌株VM15C的PVADH、偶氮螺菌属菌株EbN1的推定PVADH以及黄单胞菌属（同一性为54 - 25%）的氨基酸序列相似，并且与睾丸丛毛单胞菌、富营养罗尔斯通氏菌和恶臭假单胞菌的喹啉血红蛋白醇脱氢酶（QH - ADHs）（同一性为25 - 29%）相似。来自菌株113P3和VM15C的PVADHs具有保守的超桶结构域（SD）、SD中可能的PQQ结合氨基酸和血红素结合结构域（HBD）（它们应被指定为QH - PVADHs），但HBD和SD的氨基酸序列位置与QH - ADHs相反。提出了QH - PVADHs的蛋白质结构。斑点印迹杂交和RT - PCR结果表明，编码氧化PVA水解酶、PVADH和细胞色素c的三个基因组成型表达并形成一个操纵子。
