Collaborative Project: Metabolic Specificity and Regulation in the Methanogenic Archaea

这项研究的长期目标是了解单碳代谢的生物化学和起源。本项目的研究将确定甲基-cob酰胺的两种同工酶：辅酶M甲基转移酶（MT2-A和MT2-M）相互作用的酶学。MT2同工酶在巴氏甲烷菌的不同C-1代谢途径中起作用，其中它们催化甲基从不同的类corrinoid （B12）蛋白底物转移到辅酶m的巯基。在甲醇和三甲胺培养的巴氏甲烷菌中，这两种同工酶的相对量差异达200倍。研究旨在通过表征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。本研究的具体目的是通过对MT2基因进行工程改造，使两种MT2同工酶产生明确的结构变化，从而确定MT2同工酶-类蛋白底物特异性的结构基础。野生型和修饰的MT2蛋白将用于物理化学、动力学和热力学研究，以表征它们与不同类蛋白底物的相互作用。在这个项目完成时，负责MT2同工酶代谢特异性的蛋白质结构决定因素将被确定。对这一b12依赖性系统的研究将对理解C-1代谢特异性做出重要贡献。这个项目将进一步加深我们对代谢特异性的进化，单碳单元的酶转移，以及一种新的酶催化甲基转移机制的理解。这些酶来自产甲烷的古菌，但显然在迄今研究的所有其他形式的生命中都有类似的酶。DeMoll

DeMollThe long range goal of this research is to obtain an understanding of the biochemistry and origin of one-carbon metabolism. The research in this project will determine the enzymology of the interaction of two isozymes of methyl-cob-amide:coenzyme M methyl-transferase (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. Studies are designed 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 specific aim of this study is to identify the structural basis for MT2 isozyme-corrinoid protein substrate specificity 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, kinetic and thermodynamic studies to characterize their interactions with different corrinoid protein substrates. At the completion of this project the protein structural determinants responsible for the metabolic specificity of the MT2 isozymes will have been identified. Studies on this B12-dependent system will be an important contribution to the understanding of C-1 metabolic specificity.This project will further our understanding of the evolution of metabolic specificity, of the enzymatic transfer of single carbon units, and of a novel enzymatic zinc-based catalytic mechanism of methyl group transfer. The enzymes are derived from methanogenic Archaea, but apparently have analogous enzymatic counterparts in all other forms of life thus far studied.DeMoll