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Title: New Perspectives on Acetate and One-Carbon Metabolism in the Methanoarchaea

Abstract

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to bicarbonate. Although widespread in prokaryotes of the domains Bacteria and Archaea, few have been investigated and the physiological functions are largely unknown. Carbonic anhydrases are of biotechnological interest for carbon dioxide capture and sequestration at point sources. Prokaryotes encode three independently evolved classes. The alpha-class is restricted to a few pathogens and the other two are uniformly distributed in phylogenetically and physiologically diverse species. Although wide-spread in prokaryotes, only three gamma-class enzymes have been biochemically characterized and the physiological functions have not been investigated. The gamma-class is prominent in anaerobic acetate-utilizing methane-producing species of the genus Methanosarcina that encode three subclasses. Enzymes from two of the subclasses, Cam and CamH from Methanosarcina thermophila, have been characterized and found to utilize iron in the active site which is the first example of an iron-containing carbonic anhydrase. No representative of the third subclass has been isolated, although this subclass constitutes the great majority of the β-class. This grant application proposed to characterize gamma-class carbonic anhydrases from diverse anaerobic prokaryotes from the domains Bacteria and Archaea to broaden the understanding of this enzyme. In particular, the three subclasses present the genetically tractable acetate-utilizingmore » methanogen Methanosarcina acetivorans will be investigated to extend studies of acetate and one-carbon metabolism in this species. A genetic approach will be taken to ascertain the physiological functions. It is also proposed to delve deeper into the mechanism of Cam from M. thermophila, the archetype of the gamma-class, via a high resolution neutron structure and kinetic analysis of site-specific amino acid replacement variants. In the course of the investigation, goals were added to take advantage of discoveries on enzymes responding to oxidative stress.« less

Authors:
 [1]
  1. Pennsylvania State Univ., University Park, PA (United States)
Publication Date:
Research Org.:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1347228
Report Number(s):
DOE-PSU-20198
DOE Contract Number:
FG02-95ER20198
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Ferry, James. New Perspectives on Acetate and One-Carbon Metabolism in the Methanoarchaea. United States: N. p., 2017. Web. doi:10.2172/1347228.
Ferry, James. New Perspectives on Acetate and One-Carbon Metabolism in the Methanoarchaea. United States. doi:10.2172/1347228.
Ferry, James. Mon . "New Perspectives on Acetate and One-Carbon Metabolism in the Methanoarchaea". United States. doi:10.2172/1347228. https://www.osti.gov/servlets/purl/1347228.
@article{osti_1347228,
title = {New Perspectives on Acetate and One-Carbon Metabolism in the Methanoarchaea},
author = {Ferry, James},
abstractNote = {Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to bicarbonate. Although widespread in prokaryotes of the domains Bacteria and Archaea, few have been investigated and the physiological functions are largely unknown. Carbonic anhydrases are of biotechnological interest for carbon dioxide capture and sequestration at point sources. Prokaryotes encode three independently evolved classes. The alpha-class is restricted to a few pathogens and the other two are uniformly distributed in phylogenetically and physiologically diverse species. Although wide-spread in prokaryotes, only three gamma-class enzymes have been biochemically characterized and the physiological functions have not been investigated. The gamma-class is prominent in anaerobic acetate-utilizing methane-producing species of the genus Methanosarcina that encode three subclasses. Enzymes from two of the subclasses, Cam and CamH from Methanosarcina thermophila, have been characterized and found to utilize iron in the active site which is the first example of an iron-containing carbonic anhydrase. No representative of the third subclass has been isolated, although this subclass constitutes the great majority of the β-class. This grant application proposed to characterize gamma-class carbonic anhydrases from diverse anaerobic prokaryotes from the domains Bacteria and Archaea to broaden the understanding of this enzyme. In particular, the three subclasses present the genetically tractable acetate-utilizing methanogen Methanosarcina acetivorans will be investigated to extend studies of acetate and one-carbon metabolism in this species. A genetic approach will be taken to ascertain the physiological functions. It is also proposed to delve deeper into the mechanism of Cam from M. thermophila, the archetype of the gamma-class, via a high resolution neutron structure and kinetic analysis of site-specific amino acid replacement variants. In the course of the investigation, goals were added to take advantage of discoveries on enzymes responding to oxidative stress.},
doi = {10.2172/1347228},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Mon Mar 20 00:00:00 EDT 2017},
month = {Mon Mar 20 00:00:00 EDT 2017}
}

Technical Report:

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  • The project focused on determining the biochemical pathway and mechanisms of acetate conversion to methane in methanogenic bacteria. A corrinoid cofactor was discovered in the carbon monoxide dehydrogenase complex previously shown to be involved in the pathway of acetate conversion to methane. The genetic control mechanism for the regulation of formate dehydrogenase synthesis was determined. Results show that the enzyme is autoregulated at the level of transcription.
  • The general pathway of acetate conversion to methane in Methanosarcina thermophila is known and several of the enzymes have been characterized. AcetylCoA is cleaved by a enzyme complex with carbon monoxide dehydrogenases activity. The methyl group is transferred to Coenzyme M and the carbonyl to the nickel site in the complex. Methylcoenzyme M is reductively demethylated to methane with electrons derived from the oxidation of the bound carbonyl. A ferredoxin is the immediate electron acceptor of electrons leaving the complex. Membrane components are implicated in electron transport from the ferredoxin to methylcoenzyme m. A corrinoid protein in the complex ismore » thought to catalyze methyl transfer, electron transfer, or both. All of the above proteins have been purified except the corrinoid protein. Our laboratory is investigating the catalytic mechanism of the purified proteins. Biochemical characterizations of all proteins will include amino acid analysis and N-terminal sequencing, kinetic parameters, physical and other properties. 2 tabs.« less
  • Several enzymes in the pathway of acetate conversion to methane and carbon dioxide have been purified from Methanosarcina thermophila. The mechanisms of these enzymes are under investigation utilizing biochemical, biophysical and molecular genetic approaches. Acetate kinase and phosphotransacetylase catalyzes the activation of acetate to acetyl-CoA. The primary structure of these enzymes will be determined through cloning and sequencing of the genes. Two protein components of the CO dehydrogenase complex are under investigations. The metal centers of each component have been characterized using EPR. Cloning and sequencing of the genes for the two subunits of each component is in progress. Resultsmore » indicate that the Ni/Fe-S component cleaves the C-C and C-S bonds of acetyl-CoA followed by oxidation of the carbonyl group to carbon dioxide and transfer of the methyl group to the Co/Fe-S component. The enzymes and cofactors involved in transfer of the methyl group from the Co/Fe-S component to coenzyme M will be purified and characterized. Ferredoxin is an electron acceptor for the Ni/Fe-S component and also serves to reductively reactivate methylreductase which catalyzes the demethylation of methyl coenzyme M to methane. This ferredoxin is being characterized utilizing EPR and RR spectroscopic methods to determine the properties of the Fe-S centers. Genes encoding this and other ferredoxins have been cloned and sequenced to determine the primary structures. Carbonic anhydrase is being purified and characterized to determine the function of this enzyme in the pathway.« less
  • The following major advances were made in the past two years: both acetate activating enzymes were characterized and the regulation of synthesis was studied; direct biochemical evidence for the proposed function of the CODH was obtained, and the CODH complex was further characterized; an electron transport chain was reconstituted with CODH, ferredoxin, and membrane components that catalyzed oxidation of carbon monoxide and evolution of hydrogen; the ferredoxin was characterized; five new enzyme activities were discovered with potential for involvement in growth and/or methanogenesis from acetate; probes were developed for isolation of the genes encoding the principle enzymes of the pathway.more » 6 refs., 3 figs.« less
  • Our current understanding of the pathway of acetate conversion to methane in Methanosarcina thermophila is depicted. Our accomplishments this past year include phosphotransacetylase, the corrinoid/Fe-S protein component of the carbon monoxide dehydrogenase, and ferredoxin were characterized in further detail; two methyl transferase activities were resolved; and putative positive clones containing the phosphotransacetylase gene were isolated from a lambda gt 11 DNA library of M. thermophila. The phosphotransacetylase was purified to electrophoretic homogeneity and characterized. The specific activity was 2555 micromoles of acetyl-CoA/min/mg. Optimum activity is obtained between 35-45 /degree/C and pH 6.5-7.5 in the presence of potassium or ammonium ions(ca.more » 100 mM). The enzyme is purified as a monomer of Mr 43,000. The Km and Vmax for CoASH and acetyl phosphate are 91 and 165 micromolar, and 5517 and 4025 micromoles/min/mg. Anti-phosphotransacetylase antibodies were raised in rabbits which were used in an immunoblot of crude extracts from acetate- or methanol-grown cells; the results indicate regulation of synthesis in response to the growth substrate. A mixed oligonucleotide probe was synthesized using the DNA sequence deduced from the N-terminal amino acid sequence. 6 figs.« less