苏云金芽胞杆菌γ-氨基丁酸代谢途径相关功能基因的克隆、表达及同源性分析

Cloning, Expression and Phylogenetic Analysis of Two GABA Shunt-related Proteins from Bacillus thuringiensis

摘要:

γ-氨基丁酸代谢旁路作为三羧酸循环的一个分支，在真核、原核生物中广泛存在。在这条代谢途径中，涉及γ-氨基丁酸分解代谢的主要有两种酶：一种是γ-氨基丁酸转氨酶，能将γ-氨基丁酸转变成琥珀酸半醛；另一种是琥珀酸半醛脱氢酶，该酶能将琥珀酸半醛氧化形成琥珀酸，后者进入三羧酸循环。从国内分离得到的苏云金芽胞杆菌G03菌株中克隆了gabT 和 gabD基因。其中gabT基因含有1440 bp，编码一个大小为52.6 kD的蛋白质，而gabD 基因大小为1449 bp，编码一个 52.2 kD的蛋白质。这两个基因都分别在大肠杆菌中进行了表达和纯化。通过酶活测定结果表明，GabT和GabD蛋白分别呈现出γ-氨基丁酸转氨酶和琥珀酸半醛脱氢酶的活性。氨基酸序列同源性比对分析发现，这两个蛋白质在蜡样芽胞杆菌群(B.cereus group)中具有较高的相似性，而与枯草芽胞杆菌的相似性较低则分别为58%、51%。为进一步深入研究γ-氨基丁酸代谢旁路在苏云金芽胞杆菌中的生物学功能及其转录调控机制奠定了基础。

Abstract:

The γ-aminobutyric acid (GABA) shunt is a metabolic pathway that bypasses two successive steps of the tricarboxylic acid cycle (TCA cycle) in both prokaryotes and eukaryotes. In this pathway, the enzymes involved in GABA catabolism are GABA transaminase (GABASE), which converts GABA to succinic semialdhyde, and succinic-semialdehyde dehydrogenase (SSADH), which oxidizes succinic semialdhyde to succinate. In this report, we characterized gabT and gabD genes cloned from B.thuringiensis strain G03 isolated in China. Both gabT gene, 1440 bp encoding a protein with 52.6 kD, and gabD gene, 1449 bp encoding a protein with 52.2 kD were expressed in E.coli BL21(DE3) strain, and their products were purified by affinity chromatography. By enzyme assay,GabT protein showed the activity of GABASE, while GabD protein exhibited SSADH activity. The amino acid sequences of two proteins, GABASE and SSADH, showed significant identity with those in the B.cereus group, but their similarity score between G03 and B.subtilis 168 was lower, only 58% and 51%, respectively. In the present study, we provided evidence to identify the genes involved in GABA metabolism, and it will be helpful for further assessment the biological function(s) and regulation mechanism of GABA shunt in B.thuringiensis.