The Structure and Function of Component B of The Methylcoenzyme M Methylreductase System
Noll, Kenneth Martin
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https://hdl.handle.net/2142/71185
Description
Title
The Structure and Function of Component B of The Methylcoenzyme M Methylreductase System
Author(s)
Noll, Kenneth Martin
Issue Date
1987
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The structure and function of a novel cofactor required for in vitro methanogenesis by the methylcoenzyme M methylreductase system were investigated. When the methylreductase system was first fractionated using cell extracts from Methanobacterium thermoautotrophicum strain $\Delta$H, three fractions were found to be required. Two of these fractions, components A and C, were found to be protein components. The third, component B, was described as a low-molecular weight, heat-stable cofactor unique to methanogenic bacteria.
Component B was purified to homogeneity and its structure determined. From one kilogram of wet cells, 9 mg of pure cofactor could be obtained using a series of chromatographic steps carried out under anaerobic and aerobic conditions. Chemical analysis, $\sp{1}{\rm H}$ NMR, $\sp {\rm C}$ NMR, IR spectroscopy, and both low- and high-resolution mass spectrometry were used to elucidate the structure of the compound. From these analyses, the structure of component B was proposed to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP). This structure was confirmed by the total synthesis of the cofactor. The synthetic cofactor was identical to the authentic cofactor based upon data from instrumental analyses and its ability to stimulate methane formation in vitro using cell extracts dependent upon component B for activity.
The function of the cofactor in methanogenesis was also studied. HS-HTP was found to be tightly bound to component C. The methyl thioether derivative of HS-HTP was synthesized and found to be unable to act as a methyl-donor to the methylreductase system. This derivative inhibited methanogenesis. An adenylated form of HS-HTP appeared not to be the active form of the cofactor since methane formation in the absence of ATP was observed in a reaction that required HS-HTP. HS-HTP was capable of acting as the sole source of reducing potential for methanogenesis in a reaction carried out under a nitrogen atmosphere. Therefore, HS-HTP appears to function as the ultimate reductant in the methylcoenzyme M methylreductase system.
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