Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae
Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae
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DOI：
10.1128/aem.71.7.3858-3865.2005
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发表时间：
2005-07-01
影响因子：
4.4
通讯作者：
Lovley, DR
中科院分区：
文献类型：
--
作者：
Bond, DR;Mester, T;Lovley, DR
土杆菌科的成员通常是沉积环境中以及能量收集电极表面上的主要Fe（III）还原微生物，并且能够有效地将乙酸盐的氧化与外部电子受体的还原偶联。这些生物的柠檬酸合酶活性由于其在乙酸代谢中的关键作用而受到关注。先前的基因组测序的硫还原Geetrachylsulfreducens揭示了一个推定的柠檬酸合酶序列相关的柠檬酸脱氢酶的真核生物。柠檬酸合成酶活性在G.硫还原菌可以通过亲和层析分离为单一的49-kDa蛋白质。该酶在大肠杆菌中成功地以高水平表达，具有与天然酶相似的性质，动力学参数与相关的柠檬酸脱氢酶相当（k（cat）= 8.3 s（-1）;乙酰辅酶A和草酰乙酸的Km分别为14.1和4.3 μ M）。该酶是二聚体的，并且被ATP轻微抑制（对于乙酰辅酶A，Ki = 1.9mM），ATP是许多真核二聚体柠檬酸脱氢酶的已知抑制剂。原核六聚体柠檬酸脱氢酶的别构抑制剂NADH不影响酶活性。与大多数原核二聚体柠檬酸脱氢酶不同，该酶不具有任何甲基柠檬酸合酶活性。与真核生物和原核生物的柠檬酸脱氢酶相比，该酶的一个独特特征是缺乏K+离子的刺激。类似的柠檬酸合酶序列中检测到的其他土杆菌科成员的多样性。这第一次表征的真核生物样柠檬酸合酶从原核生物提供了新的见解乙酸盐代谢的Geobacteraceae成员，并提出了一个分子目标，用于跟踪这些生物体在环境中的存在和活动。
Members of the family Geobacteraceae are commonly the predominant Fe (III) -reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (k(cat) = 8.3 s(-1); K-m = 14.1 and 4.3 mu M for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K-i = 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.