Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila

Abstract

The cell extract protein content of acetate-and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate-and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate-and methanol-grown Methanosarcina thermnophila. Levels of formyltransferase in either acetate-or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate-and methanol-grown Methanosarcina thermophila.Of the known methanogens, Methanosarcina species are the most catabolically diverse and are therefore amenable to the study of catabolite influence on protein synthesis. Methanosarcina thermophila TM-1 is capable of using acetate, methanol, or methylated amines as growth substrates (28). Except for the reductive demethylation of methyl coenzyme M, the pathways for utilization of acetate or methanol by the methanogenic archaeobacteria (see Fig. 3) (23, 25) are distinct from the pathway for reduction of carbon dioxide (20). When acetate is used, acetate kinase and phosphotransacetylase catalyze the activation of acetate to acetyl coenzyme A prior to the proposed cleavage of the carbon-carbon bond by the carbon monoxide dehydrogenase (CODH) complex (23). The levels of these enzymes are severalfold greater in acetate-grown cells than they are in methanol-grown cells (1,15,23). Electrons for the demethylation of methyl coenzyme M derive from the oxidation of methanol (see reaction 5 in Fig. 3) or the carbonyl group of acetate (see reaction 2 in Fig. 3). The presence of formylmethanofuran dehydrogenase activity in extracts of methanol-grown Methanosarcina barkeri is consistent with the proposal that methanol is oxidized by a reversal of the carbon dioxide reduction pathway (5). Growth of Methanosarcina thermophila TM-1 is supported by acetate or methanol, but the organism reduces carbon dioxide to methane only after an extended adaptation period, and growth is poor (28). Here we present results of two-dimensional (2-D) gel electrophoretic analyses of whole-cell proteins which estimate the extent of regulation by the growth substrate and report the activities of the enzymes that catalyze one-carbon redox reactions in extracts of acetate-and methanol-grown cells.

Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein

“…In dant enzymes of glycolysis and fermentation. In many cases, these abundant proteins formed charge chains or exhibited microheterogeneity analogous to abundant proteins in other systems (20,39,43). Loading the isoelectric focussing dimension from the acidic end would be expected to minimize or alter chemical reactions and protein associations.…”

Methane from acetate

“…(AGO' = -103 kJ/mol CH4 [55]) (9) Thus, cells grown with a mixture of these substrates utilize methanol first. Two-dimensional gel electrophoresis of cell extract proteins from M. thermophila reveal more than 100 mutually exclusive peptides in acetate-and methanol-grown cells (22). This regulation of enzyme synthesis in response to the growth substrate presents an opportunity for future investigations into the molecular basis for transcriptional regulation in members of the Archaea domain.…”

“…Growth of M. barkeri and M. thermophila on acetate induces carbonic anhydrase activity, but the function of this enzyme in the conversion of acetate to methane is unknown (22,33). It is proposed that the formation of carbonic acid may be required in an antiport mechanism for transport of the acetate anion into the cell (33).…”

“…Acetate-grown Methanosarcina species contain low levels of enzymes which are utilized in the C02-reduction pathway for methanogenesis; thus, it is proposed that the methyl group of acetate is oxidized by a reversal of the pathway for CO2 reduction to methane (22). Acetate-grown M. barkeri contains formyl-methanofuran dehydrogenase and 5,10-methylene-H4MPT (tetrahydromethanopterin) dehydrogenase (47) and acetate-grown M. thermophila contains formyl-methanofuran:H4MPT formyltransferase, 5,10-methenyl-H4MPT+ cyclohydrolase, and F420-dependent 5,10-methylene-H4MPT dehydrogenase (22). The low levels of these enzymes suggest they are not involved in conversion of the methyl group of acetate to methane, but in oxidation of the methyl group of acetate to CO2.…”

Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila

“…In a reexamination of the C. kluyveri Pta workers attempted to detect an acetyl-enzyme intermediate; however, no isotope exchange from labeled acetyl phosphate into either acetyl-CoA or inorganic phosphate was observed in the absence of free CoA, and attempts to isolate an acetyl-Pta intermediate were unsuccessful, which is consistent with a ternary complex mechanism (9). Although numerous genetic and physiological studies have continued to demonstrate the universal function of Pta in acetate metabolism in diverse microbes (1,4,6,8,15,17,24,25,27,30,32,38), mechanistic analyses of the enzyme were abandoned until there was an investigation of Pta from the archaeon Methanosarcina thermophila, which obtains energy for growth by converting acetate to methane (22).Cloning of the gene and heterologous expression of Pta from M. thermophila allowed the large-scale production of protein required for structural studies, biochemical analyses, and the use of site-specific replacement to analyze the function of specific residues (22). Cys 312 was predicted to be present in the active site, although it is not essential for catalysis (31).…”