Identification and Characterization of a Re-Citrate Synthase in Dehalococcoides Strain CBDB1
Identification and Characterization of a Re-Citrate Synthase in Dehalococcoides Strain CBDB1
复制标题
DOI：
10.1128/jb.05120-11
复制
发表时间：
2011-10-01
影响因子：
3.2
通讯作者：
Adrian, Lorenz
中科院分区：
文献类型：
--
作者：
Marco-Urrea, Ernest;Paul, Steffanie;Adrian, Lorenz
所有已测序的脱氢球菌菌株的基因组注释都缺乏柠檬酸合成酶，尽管生理实验表明应该对这种活性进行编码。本文报道了由嗜盐球菌属菌株CBDB1合成的一种表面特异性柠檬酸合成酶，该功能由cbdbA1708(NCBI登录号CAI83711)基因编码，以前被注释为编码同型柠檬酸合成酶。在大肠杆菌中异源表达了cbdbA1708基因，并对重组酶进行了纯化。该酶催化草酰乙酸酯和乙酰辅酶A(乙酰辅酶A)缩合成柠檬酸。该蛋白不具有同位柠檬酸合成酶活性，并被柠檬酸抑制，需要Mn2+才能充分发挥活性。用电喷雾电离液质联用(ESI LC/MS)检测异源表达的柠檬酸合成酶的立体特异性。以[2-C-13]乙酰辅酶A和草酰乙酸酯为原料，用脱卤球菌类重组柠檬酸合成酶催化合成柠檬酸，再用市售柠檬酸裂解酶加苹果酸脱氢酶催化合成乙酸酯和苹果酸。未标记的醋酸酯和C-13标记的苹果酸的形成证明了该酶的表面特异性活性。对菌株CBDB1细胞提取液的蛋白质组学分析表明，菌株CBDB1中表达了cbdbA1708。
The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-C-13] acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and C-13-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.