Collaborative Project: Metabolic Specificity and Regulation in the Methanogenic Archaea

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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