CO DEHYDROGENASE AND ACETOCLASTIC METHANOGENESIS IN METHANOSARCINA BARKERI

巴克甲烷八球菌中的 CO 脱氢酶和乙酰分解产甲烷作用

• 批准号: 3919979
• 负责人: D A GRAHAME
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3919979
• 关键词: Methanobacteriaceae; acetates; acetyl coA; adenosine triphosphate; carbon monoxide; chelating agents; covalent bond; enzyme mechanism; enzyme structure; iron; methanol; methyl group; microorganism culture; microorganism metabolism; nickel; tissue /cell culture; transmethylation

In experiments with Methanosarcina barkeri it was determined that an intact and energized cell membrane is required for carbon-carbon bond cleavage of acetate as measured by isotopic exchange of carbon dioxide. Although extensive isotopic exchange was not detected in soluble preparations, methanogenesis and methyl-SCoM formation from acetate were readily observed in the presence of ATP and hydrogen. The rate of in vitro methanogenesis was dependent upon both ATP and acetate concentrations. The lag before onset of methanogenesis was not due to a slow production of methyl-SCoM, but rather to activation of step(s) needed for reduction of methyl-SCoM to HSCoM and methane. Both processes of methanogenesis and methyl-SCoM formation from acetate were stimulated by coenzyme A which indicates that acetyl-CoA is the activated form of acetate required for cleavage in methanogenesis. A convenient method was developed for analysis of the enzyme responsible for formation of methyl-SCoM from a methylated corrinoid compound and HSCoM. Cells of M. barkeri grown on acetate exhibited essentially the same level of this enzyme as cells grown on methanol. This suggests a common methyl group transfer step in both pathways of acetate and methanol metabolism. The methyltransferase enzyme was purified to near homogeneity and was found to be monomeric with a molecular weight of 37,000. It showed a high affinity for HSCoM (Km = 0.086 mM), but weakly bound methylcobalamin (Km = 13.5 mM). Metal ion chelators inhibited the enzyme but reactivation was brought about by removal of the chelator or addition of divalent metal ions. Supplementation of extracts producing methane and methyl-SCoM from acetate with purified carbon monoxide dehydrogenase (CODH) and the methyltransferase, respectively, failed to stimulate these processes. This suggests that the levels of CODH and methyltransferase were saturating under the conditions tested and that another unidentified step(s) was predominantly rate limiting.

在巴氏甲烷八叠球菌的实验中，确定了 碳-碳需要一个完整的充满能量的细胞膜 通过碳的同位素交换测量的醋酸盐键断裂 二氧化物 虽然在这些实验中没有检测到广泛的同位素交换， 可溶性制剂、产甲烷和甲基-SCoM形成 在ATP和氢的存在下容易观察到乙酸盐。 体外产甲烷速率依赖于ATP和 乙酸盐浓度。 甲烷生成开始前的滞后是 不是由于甲基-SCoM的缓慢产生，而是由于 激活甲基-SCoM还原为HSCoM所需的步骤 和甲烷。 甲烷生成和甲基-SCoM的两个过程 辅酶A刺激乙酸盐的形成， 表明乙酰辅酶A是所需的乙酸盐的活化形式 用于甲烷生成中的裂解。 开发了一种简便的方法 用于分析负责甲基-SCoM形成的酶 从甲基化的类可啉化合物和HSCoM。 M.细胞 在乙酸盐上生长的巴氏杆菌表现出基本上相同水平的 这种酶是在甲醇中生长的细胞。 这表明， 乙酸和甲醇两种途径中的甲基转移步骤 新陈代谢. 将甲基转移酶纯化至接近 均匀性，并被发现是单体的分子量 37000人。 它显示出对HSCoM的高亲和力（Km = 0.086 mM）， 但弱结合的甲钴胺（Km = 13.5mM）。 金属离子 螯合剂抑制了酶的活性，但使酶重新活化 通过除去螯合剂或加入二价金属离子。 补充从植物中产生甲烷和甲基-SCoM的提取物 乙酸与纯化的一氧化碳脱氢酶（CODH）和 甲基转移酶，分别，未能刺激这些 流程. 这表明，CODH和 甲基转移酶在测试条件下饱和， 另一个未识别的步骤主要是速率限制。