Collaborative Project: Metabolic Specificity and Regulation in the Methanogenic Archaea

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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.