Kanwal Simab, Incharoensakdi Aran
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Plant Physiol Biochem. 2016 Feb;99:59-65. doi: 10.1016/j.plaphy.2015.12.008. Epub 2015 Dec 18.
Glutamate decarboxylase (GAD) (EC 4.1.1.15), an enzyme responsible for the synthesis of γ-aminobutyric acid (GABA), from Synechocystis sp. PCC6803 was cloned and overexpressed in Escherichia coli BL21(DE3). The purified enzyme was expressed as a monomeric protein with a molecular mass of 53 and 55 kDa as determined by SDS-PAGE and gel filtration chromatography, respectively. The enzyme activity was pyridoxal-5'-phosphate dependent with an optimal activity at pH 6.0 and 30 °C. The catalytic properties of this enzyme were, Km = 19.6 mM; kcat = 100.7 s(-1); and kcat/Km = 5.1 mM(-1) s(-1). The transcription levels of genes involved in nitrogen metabolism were up-regulated in the Δgad strain. The mutant showed approximately 4- and 8-fold increases in the transcript levels of kgd and gabdh encoding a novel α-ketoglutarate decarboxylase and γ-aminobutanal dehydrogenase, respectively. Overall results suggested that in Synechocystis lacking a functional GAD, the γ-aminobutanal dehydrogenase might serve as an alternative catalytic pathway for GABA synthesis.
来自集胞藻PCC6803的谷氨酸脱羧酶（GAD）（EC 4.1.1.15）是一种负责合成γ-氨基丁酸（GABA）的酶，该酶被克隆并在大肠杆菌BL21(DE3)中过表达。经SDS-PAGE和凝胶过滤色谱法测定，纯化后的酶以单体蛋白形式表达，分子量分别为53 kDa和55 kDa。该酶的活性依赖于磷酸吡哆醛，在pH 6.0和30℃时具有最佳活性。该酶的催化特性为：Km = 19.6 mM；kcat = 100.7 s(-1)；kcat/Km = 5.1 mM(-1) s(-1)。在Δgad菌株中，参与氮代谢的基因转录水平上调。该突变体中，分别编码一种新型α-酮戊二酸脱羧酶和γ-氨基丁醛脱氢酶的kgd和gabdh的转录水平分别增加了约4倍和8倍。总体结果表明，在缺乏功能性GAD的集胞藻中，γ-氨基丁醛脱氢酶可能作为GABA合成的替代催化途径。
