Biosynthesis of 7-Mercaptoheptanylthreonine-phosphate and the Methyl Reducing Factor in Mehtanogenic Bacteria

本项目将建立产甲烷辅酶7-巯基庚基苏氨酸-磷酸（HS-HTP）和甲基还原辅因子（MRF）的生物合成途径。第一阶段的工作将是确定HS-HTP和MRF在不同种类的产甲烷古细菌中的存在，并确定这些辅酶之间可能的前体/产物关系。分子的存在和相互转化将通过荧光测量它们的单溴烷衍生物来确定，使用作者最近开发的程序。将确定单个亚基和亚基分子群与HS-HTP和MRF的结合。将进行体内和体外实验，使用稳定的同位素标记分子和质谱法来测量掺入，以帮助确定生物合成途径。产甲烷古细菌将二氧化碳代谢还原为甲烷的生物化学研究代表了微生物代谢的一个令人兴奋的领域。这种代谢的一个重要方面是发现了七种新的辅酶。甲烷途径中的许多辅酶具有与其他已知辅酶和维生素完全不同的化学结构。正因为如此，每个结构都代表着一种新的生物化合物，具有不寻常的功能。在本奖项中，将探索7-巯基庚基苏氨酸-磷酸和甲基还原辅因子两种辅因子的生物合成、结构和在有限范围内的分布。需要解决的具体问题有：这些辅助因子是如何生物合成和组装的，它们之间是否存在生物合成关联？这些问题的答案可能会为我们对产甲烷辅酶的生物合成的理解完成另一章，并为甲烷这一重要能源的形成提供更多的微生物生物化学线索。＊＊＊

White 9311538 In this project, the biosynthetic pathway(s) for the methanogenic coenzymes 7-mercaptoheptanylthreonine-phosphate (HS-HTP) and methyl reducing cofactor (MRF) will be established. The first phase of work will be to establish the presence of HS-HTP and MRF in different species of methanogenic archaebacteria and to determine any possible precursor/product relationship between these coenzymes. The presence and interconversion of the molecules will be determined by fluorescence measurement of their monobromobimane derivatives using procedures recently developed by the author. The incorporation of individual subunit and groups of subunit molecules into HS-HTP and MRF will be determined. Both in vivo and in vitro experiments, using stable isotopically-labeled molecules and mass spectrometry to measure incorporation, will be performed to help define the biosynthetic pathways. %%% The biochemistry of the metabolic reduction of carbon dioxide to methane by the methanogenic archaebacteria represents an exciting area of microbiological metabolism. One important aspect of this metabolism is the discovery of seven new coenzymes. Many of the coenzymes in the methane pathway have chemical structures completely different from other well known coenzymes and vitamins. Because of this each structure represents a new biological compound with unusual functions to be discovered. In this award, the biosynthesis, structure, and to a limited extent, the distribution of two cofactors, 7-mercaptoheptanylthreonine-phosphate and methyl reducing cofactor, will be explored. Specific questions to be addressed are; how are each of these cofactors biosynthesized and assembled, and are they biosynthetically interrelated? Answers to these questions may complete another chapter in our understanding of the biosynthesis of the methanogenic coenzymes, and also shed more light on the microbial biochemistry of the formation of methane, an important energy source. ***