Collaborative Project: Metabolic Specificity and Regulation in the Methanogenic Archaea

合作项目：产甲烷古菌的代谢特异性和调控

• 批准号: 9905068
• 负责人: David Grahame
• 金额: $ 24.45万
• 依托单位: Henry M Jackson Fdn for Advmt of Military Medicine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-10-01 至 2004-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9905068&HistoricalAwards=false
• 关键词: Collaborative; Project; Metabolic; Specificity; Regulation

Grahame The long range goal of this research is to obtain a detailed understanding of the biochemistry of one-carbon metabolism. Investigations are focused on discovering how C-1 metabolism is regulated in the methanogenic Archaea, which has broad significance toward developing an understanding of how gene regulation takes place in these unusual organisms. This project specifically concerns two isozymes of methyl-cob-amide:coenzyme M methyl-transfer-ase (MT2-A and MT2-M). The MT2 isozymes function in separate C-1 metabolic pathways in Methanosarcina barkeri, in which they catalyze methyl group transfer from different corrinoid (B12) protein substrates to the thiol group of coenzyme M. The relative amounts of the two isozymes differ 200-fold in M. barkeri grown on methanol versus trimethylamine, with evidence suggesting control at the level of transcription. The research will uncover the biochemical mechanisms responsible for regulating the differential transcription of the two MT2 isozyme genes. In addition, the work will define the molecular basis for the functional specificity of MT2 isozymes in different metabolic pathways by characterizing the interactions of the MT2 isozymes with different corrinoid proteins. The structural basis for MT2 isozyme-corrinoid protein substrate specificity will be identified by engineering the MT2 genes to produce defined structural changes in both MT2 isozymes. Wild type and modified MT2 proteins will be used in physicochemical and kinetic studies to characterize their interactions with different corrinoid protein substrates. The Co-PI will carry out a collaborative study on the thermodynamics of corrinoid protein binding to the mutant and wild-type MT2 isozymes. The MT2 isozyme system will be used to investigate regulation of carbon substrate utilization pathways in the Archaea by exploring the molecular events that control the wide variation in the levels of the isozymes in response to changes in growth substrate. The focus is to identify the biochemical mechanism(s) responsible for differential expression of the two MT2 genes. Methanogens make up the largest genetically and metabolically distinct division within the Archaea (a group of unusual organisms that constitutes a fundamental domain of life separate from Bacteria and Eukaryotes). Methanogens growing on a variety of simple carbon substrates contribute approximately one-billion tons of methane to the yearly production of this gas on Earth. In every methane-generating pathway known, one or more vitamin B12-dependent reactions is required. In Methanosarcina barkeri alone there are as many as seven different B12-containing proteins involved in different methane forming pathways. Despite their metabolic importance, only recently has progress been made in studying these B12 proteins and their associated methyltransferase enzymes in purified form. At present, little is known about the molecular details of how the expression of these enzymes is regulated in response to physiological or environmental changes. Moreover, this is true in general for the mechanisms by which gene regulation takes place in the Archaea, and the means by which environmental signals bring about control of gene expression in these organisms have yet to be identified and characterized. In this work, we will gain an understanding about the specificity of B12 methyltransferase interactions in different metabolic pathways, and the mechanism by which the levels of these enzymes are differentially controlled depending on nutrient availability. The research carried out here will advance our knowledge of the structural basis for specificity in B12 protein interactions, and will play an important part in understanding the basic mechanisms of global regulation of gene expression involved in nutient utilization. It will further our knowledge of ecology and the environment, and ultimately improve our usage of the metabolic potential of the methanogenic Archaea for agricultural, biomedical, and industrial applications.

这项研究的长期目标是详细了解一碳代谢的生物化学。 调查的重点是发现C-1代谢是如何调节产甲烷菌，这对了解基因调控如何发生在这些不寻常的生物体具有广泛的意义。 本项目具体涉及甲基辅酶A的两种同工酶：辅酶M甲基转移酶（MT 2-A和MT 2-M）。 MT 2同工酶在巴氏甲烷八叠球菌的不同C-1代谢途径中起作用，其中它们催化甲基从不同的类咕啉（B12）蛋白底物转移到辅酶M的巯基。 两种同工酶的相对含量在M中相差200倍。巴氏杆菌在甲醇上生长，而不是在三甲胺上生长，有证据表明在转录水平上进行控制。 这项研究将揭示负责调节两种MT 2同工酶基因差异转录的生化机制。 此外，这项工作将定义MT 2同工酶在不同代谢途径中的功能特异性的分子基础，通过表征MT 2同工酶与不同corrinoid蛋白的相互作用。 MT 2同工酶-类咕啉蛋白底物特异性的结构基础将通过改造MT 2基因以在两种MT 2同工酶中产生确定的结构变化来鉴定。 野生型和修饰的MT 2蛋白将用于物理化学和动力学研究，以表征它们与不同的类咕啉蛋白底物的相互作用。 Co-PI将开展一项关于corrinoid蛋白与突变型和野生型MT 2同工酶结合的热力学的合作研究。 MT 2同工酶系统将被用来研究调控的碳底物利用途径在的的葡萄球菌通过探索的分子事件，控制在响应生长底物的变化的同工酶水平的广泛变化。 重点是确定负责两个MT 2基因差异表达的生化机制。 产甲烷菌是生物界中最大的遗传和代谢上不同的部门（一组不寻常的生物体，构成了与细菌和真核生物分开的基本生命领域）。 在各种简单碳基质上生长的产甲烷菌每年为地球上这种气体的产生贡献大约10亿吨甲烷。 在每一个已知的甲烷生成途径中，都需要一个或多个维生素B12依赖性反应。 仅在巴氏甲烷八叠球菌中，就有多达7种不同的含B12蛋白参与不同的甲烷形成途径。 尽管其代谢的重要性，只是最近才取得了进展，在研究这些B12蛋白质及其相关的甲基转移酶的纯化形式。 目前，人们对这些酶的表达如何响应生理或环境变化进行调节的分子细节知之甚少。 此外，这是真实的，在一般的机制，基因调控发生在微生物，以及环境信号的手段带来的基因表达的控制，在这些生物体还有待确定和特点。 在这项工作中，我们将了解B12甲基转移酶在不同代谢途径中相互作用的特异性，以及这些酶的水平根据营养物质的可用性而受到差异控制的机制。 本研究将进一步加深我们对B12蛋白相互作用特异性的结构基础的认识，并将在理解营养物质利用中基因表达的全局调控的基本机制方面发挥重要作用。 它将进一步加深我们对生态和环境的了解，并最终提高我们对产甲烷菌代谢潜力的利用，用于农业，生物医学和工业应用。