Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway
Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway
复制标题
DOI：
10.1128/aem.04146-13
复制
发表时间：
2014-04-01
影响因子：
4.4
通讯作者：
Kelly, Robert M.
中科院分区：
文献类型：
--
作者：
Hawkins, Aaron B.;Adams, Michael W. W.;Kelly, Robert M.
极端嗜热的古生菌Metallosphaera sedula(最适生长温度73℃，pH 2.0)通过3-羟基丙酸酯/4-羟基丁酸酯(3HP/4HB)碳固定循环在金属硫化物或分子氢上化学自养生长。这个循环将两个二氧化碳分子加到乙酰辅酶A(乙酰辅酶A)上生成4HB，然后重排和裂解形成两个乙酰辅酶A分子。先前的代谢流量分析表明，三分之二的中心碳前体分子来自琥珀酰辅酶A，它被氧化成苹果酸和草酰乙酸酯。剩下的三分之一显然是来自乙酰辅酶A。因此，超越琥珀酰辅酶A的步骤对于完成碳固定循环和乙酰辅酶A的消退是必不可少的。在此，3HP/4HB循环的最后四种酶，4-羟丁酸-辅酶A连接酶(MSED_0406)，4-羟丁酰-辅酶A脱水酶(MSED_1321)，巴豆酰-辅酶A水合酶/(S)-3-羟丁酰-辅酶A脱氢酶(MSED_0399)和乙酰乙酰-辅酶Aβ-酮硫醇酶(MSED_0656)，在体外组合在大肠杆菌中被重组产生，并被证明能将4HB转化为乙酰辅酶A。在自养(CO2限制)和异养条件下，利用气体密集型生物反应器系统研究了连接CO2固定和中心代谢的代谢途径。转录分析表明，3HP/4HB途径在向合成代谢途径供应乙酰辅酶A方面具有重要作用，这些途径产生了小草杆菌代谢中的中间产物。结果表明，3HP/4HB途径中琥珀酸支和乙酰辅酶A分支之间的通量由4-羟丁酸-辅酶A连接酶控制，可能受蛋白质乙酰转移酶(PAT)/Sir2依赖的翻译后系统的调节。综上所述，这项工作证实了3HP/4HB途径的最后四个步骤，从而为研究CO2固定和七叶草中枢代谢之间的联系提供了框架。
The extremely thermoacidophilic archaeon Metallosphaera sedula (optimum growth temperature, 73 degrees C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2 molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA beta-ketothiolase (Msed_0656), were produced recombinantly in Escherichia coli, combined in vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2 fixation and central metabolism were examined using a gas-intensive bioreactor system in which M. sedula was grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates in M. sedula metabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2 fixation and central metabolism in M. sedula.