Takanashi Masahiko, Shiraki Mari, Saito Terumi
Laboratory of Molecular Microbiology, Department of Biological Science, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa, Japan.
Antonie Van Leeuwenhoek. 2009 Mar;95(3):249-62. doi: 10.1007/s10482-009-9308-9. Epub 2009 Feb 15.
We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.
我们之前报道过，当在细胞外聚-3-羟基丁酸酯（PHB）上生长的皮氏罗尔斯通氏菌T1在不同碳源（如3-羟基丁酸酯（3HB）和琥珀酸）上生长时，两种3-羟基丁酸脱氢酶（BDH1和BDH2）的活性会受到培养条件的极大影响。在本研究中，构建了bdh1或bdh2的敲除突变体，并在不同培养条件下对其进行了表征。此外，在bdh2突变体中发现了一种新的BDH（BDH3），并对bdh3进行了克隆。BDH3底物的表观动力学参数表明，该酶适用于3-羟基丁酸酯（3HB）氧化为乙酰乙酸的反应。在蛋白质免疫印迹分析中，很明显BDH3仅在以3HB或PHB作为碳源生长的细胞中产生，而BDH1和BDH2在以各种碳源（如糖类、氨基酸、有机酸、3HB和PHB）生长的细胞中产生。当细胞用3HB、柠檬酸盐、琥珀酸盐或营养肉汤培养时，bdh1和bdh2突变体的生长速率均落后于野生型。对作为氧化应激的二酰胺的敏感性测试表明，缺乏BDH1或BDH2会导致中和应激的能力下降。这些结果表明，BDH1和BDH2对于调节细胞质氧化还原状态以及利用3HB是必需的，而BDH3专门用于利用3HB。bdh3的表达可能与编码假定的3HB通透酶的基因协同调控。
