School of Life Sciences (KNU Creative BioResearch Group), KNU Institute for Microorganisms, Kyungpook National University, Daegu, 41566, Republic of Korea.
School of Life Sciences (KNU Creative BioResearch Group), KNU Institute for Microorganisms, Kyungpook National University, Daegu, 41566, Republic of Korea.
Biochem Biophys Res Commun. 2019 Feb 12;509(3):722-727. doi: 10.1016/j.bbrc.2018.12.172. Epub 2019 Jan 3.
Metallosphaera sedula is a thermoacidophilic archaeon that has carbon fixation ability using the 3-hydroxypropionate/4-hydroxybutyrate(3-HP/4-HB) cycle, and has an incomplete TCA cycle to produce necessary biosynthetic precursors. The citrate synthase from M. sedula (MsCS) is an enzyme involved in the first step of the incomplete TCA cycle, catalyzing the conversion of oxaloacetate and acetyl-CoA into citrate and coenzyme A. To investigate the molecular mechanism of MsCS, we determined its crystal structure at 1.8 Å resolution. As other known CSs, MsCS functions as a dimer, and each monomer consists of two domains, a large domain and a small domain. We also determined the structure of the complex with acetyl-CoA and revealed the acetyl-CoA binding mode of MsCS. Structural comparison of MsCS with another CS in complex with oxaloacetate enabled us to predict the oxaloacetate binding site. Moreover, we performed inhibitory kinetic analyses of MsCS, and showed that the protein is inhibited by citrate and ATP by competitive and non-competitive inhibition modes, respectively, but not by NADH. Based on these results, we suggest that MsCS belongs to the type-I CS with structural and biochemical properties similar to those of CSs involved in the conventional TCA cycle.
喜温硫杆菌是一种嗜热嗜酸古菌，具有利用 3-羟基丙酸/4-羟基丁酸（3-HP/4-HB）循环固定碳的能力，并且具有不完全三羧酸（TCA）循环来产生必要的生物合成前体。来自喜温硫杆菌（M. sedula）的柠檬酸合酶（MsCS）是参与不完全 TCA 循环第一步的酶，催化草酰乙酸和乙酰辅酶 A 转化为柠檬酸和辅酶 A。为了研究 MsCS 的分子机制，我们测定了其在 1.8Å分辨率下的晶体结构。与其他已知的 CS 一样，MsCS 作为二聚体发挥作用，每个单体由两个结构域组成，一个大结构域和一个小结构域。我们还测定了与乙酰辅酶 A 的复合物的结构，并揭示了 MsCS 的乙酰辅酶 A 结合模式。与另一个与草酰乙酸结合的 CS 的结构比较使我们能够预测草酰乙酸的结合位点。此外，我们对 MsCS 进行了抑制动力学分析，结果表明该蛋白分别通过竞争性和非竞争性抑制模式被柠檬酸和 ATP 抑制，但不受 NADH 抑制。基于这些结果，我们认为 MsCS 属于具有与参与传统 TCA 循环的 CS 相似的结构和生化特性的 I 型 CS。
