Abe Tomoko, Kobayashi Teruyuki, Saito Terumi
Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
J Bacteriol. 2005 Oct;187(20):6982-90. doi: 10.1128/JB.187.20.6982-6990.2005.
A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.
我们鉴定出了真养产碱菌（以前称为嗜麦芽窄食单胞菌）H16的一种新型细胞内聚（3-羟基丁酸酯）（PHB）解聚酶（PhaZd），它与皮氏产碱菌T1的细胞外PHB解聚酶的催化结构域具有相似性。从核苷酸序列推导的PhaZd氨基酸序列中催化三联体（Ser190-Asp266-His330）和氧阴离子洞（His108）的位置与皮氏产碱菌T1的细胞外PHB解聚酶大致相符，但缺少信号肽、连接结构域和底物结合结构域。克隆了PhaZd基因，并从大肠杆菌中纯化了基因产物。PhaZd对人工无定形PHB颗粒的比活性明显高于真养产碱菌H16的其他已知细胞内PHB解聚酶或3-羟基丁酸酯（3HB）寡聚物水解酶。该酶降解人工无定形PHB颗粒并主要释放各种3-羟基丁酸酯寡聚物。PhaZd在PHB包涵体和胞质部分之间几乎平均分布。在各种培养条件下，phaZd缺失突变体细胞中的PHB含量高于野生型细胞。这些结果表明，PhaZd是一种新型细胞内PHB解聚酶，它与其他PHB解聚酶一起参与真养产碱菌H16中PHB的动员。
