Collaborative Project: Metabolic Specificity and Regulation in the Methanogenic Archaea

这项研究的长期目标是获得对单碳代谢的生物化学的详细了解。研究的重点是发现C-1代谢是如何在产甲烷的古菌中被调节的，这对于了解这些不寻常的生物中基因调控是如何发生的具有广泛的意义。本项目特别关注甲基cob酰胺的两种同工酶：辅酶M甲基转移酶（MT2-A和MT2-M）。在barkeri中，MT2同工酶在不同的C-1代谢途径中起作用，其中它们催化甲基从不同的类corrinoid （B12）蛋白底物转移到辅酶m的巯基。在甲醇和三甲胺中生长的barkeri中，这两种同工酶的相对数量差异200倍，有证据表明在转录水平上有控制。该研究将揭示负责调节两种MT2同工酶基因差异转录的生化机制。此外，该工作将通过表征MT2同工酶与不同类角质蛋白的相互作用，确定MT2同工酶在不同代谢途径中功能特异性的分子基础。MT2同工酶-类角质蛋白底物特异性的结构基础将通过工程MT2基因在两种MT2同工酶中产生明确的结构变化来确定。野生型和修饰的MT2蛋白将用于物理化学和动力学研究，以表征它们与不同类蛋白底物的相互作用。Co-PI将对类蛋白与突变型和野生型MT2同工酶结合的热力学进行合作研究。MT2同工酶系统将用于研究古细菌碳底物利用途径的调节，通过探索控制同工酶水平随生长底物变化的分子事件。重点是确定负责两个MT2基因差异表达的生化机制。产甲烷菌构成了古细菌（一组不同寻常的生物，构成了与细菌和真核生物分离的基本生命领域）中最大的遗传和代谢独特的分支。生长在各种简单碳基质上的产甲烷菌每年在地球上产生大约10亿吨甲烷。在每一个已知的甲烷生成途径中，都需要一个或多个依赖维生素b12的反应。仅在barkeri甲烷菌中，就有多达7种不同的含有b12的蛋白质参与不同的甲烷形成途径。尽管它们具有重要的代谢作用，但直到最近才在研究这些B12蛋白及其纯化形式的相关甲基转移酶方面取得进展。目前，对于这些酶的表达是如何响应生理或环境变化而被调节的分子细节知之甚少。此外，对于古细菌中基因调控发生的一般机制，以及环境信号控制这些生物中基因表达的手段，这是正确的，但尚未被识别和表征。在这项工作中，我们将了解不同代谢途径中B12甲基转移酶相互作用的特异性，以及这些酶的水平根据营养可用性受到不同控制的机制。本研究将促进我们对B12蛋白相互作用特异性的结构基础的认识，并将在理解涉及营养利用的基因表达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认识，并最终提高我们对产甲烷古菌在农业、生物医学和工业应用中的代谢潜力的利用。

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.