University of Illinois Urbana-Champaign

Biochemical and molecular characterization of tetrahydromethanopterin-dependent enzymes in methanogenesis

DiMarco, Anthony Andrew

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Description

Title
Biochemical and molecular characterization of tetrahydromethanopterin-dependent enzymes in methanogenesis
Author(s)
DiMarco, Anthony Andrew
Issue Date
1990
Doctoral Committee Chair(s)
Wolfe, R.S.
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, Molecular
  • Biology, Microbiology
Language
eng
Abstract
  • The biosynthetic pathway of methanogenesis proceeds via several bound intermediates. One of the coenzymes involved in this conversion is tetrahydromethanopterin (H$\sb4$MPT), a structural and biochemical analog of tetrahydrofolate (THF). Enzymes mediating these reactions in Methanobacterium thermoautotrophicum were subjected to biochemical and molecular analysis to determine their relatedness to the analogous THF-dependent reactions in non-methanogens.
  • Purification of the 5,10-methenyl-H4MPT cyclohydrolase (CYC) revealed that this enzyme was distinct and separable from the enzymes catalyzing the previous formyl group transfer reaction and subsequent reduction reaction. This is an important evolutionary consideration with respect to the THF-utilizing enzymes. Extensive biochemical analysis of the cyclohydrolase was performed to characterize the enzyme's analogy to its THF-utilizing counterpart. This work suggested a functional conservation within the pterin binding sites of different pterin enzymes. To investigate this further, the gene encoding the formyl-MFR:H$\sb4$MPT formyltransferase (FTR) was cloned and sequenced. No similarity with other pterin enzymes was found; FTR apparently possesses a novel pterin binding site.
  • FTR was expressed as a catalytically functional enzyme in E. coli. As the first such success with a methane biosynthetic enzyme, this is an encouraging invitation to further manipulations. Surprisingly, ftr is transcribed as a monocistronic message; promoter and terminator sequences were identified.
  • Biochemical analysis of methyltransfer reactions in this organism, compounded with additional observations relating to other enzymes involved in methanogenesis, led to the proposition of a methanogenic metabolon, the methanogenosome, where methanogenic reactions are physically integrated, but separated from enzyme reactions not involved in methanogenesis.
Type of Resource
text
Permalink
http://hdl.handle.net/2142/22538
Copyright and License Information
Copyright 1990 DiMarco, Anthony Andrew

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Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Microbiology