Investigation of serine hydroxymethyltransferase in methanogens

1998 ◽  
Vol 44 (7) ◽  
pp. 652-656 ◽  
Author(s):  
Zhaosheng Lin ◽  
Richard Sparling
Keyword(s):  
Key Words ◽  
Pyridoxal Phosphate ◽  
Methanobacterium Thermoautotrophicum ◽  
Methanosarcina Barkeri ◽  
Serine Hydroxymethyltransferase ◽  
Methanococcus Thermolithotrophicus ◽  
Cofactor Specificity ◽  
Methanospirillum Hungatei ◽  
Methanosphaera Stadtmanae ◽  
Methanosaeta Concilii

The cofactor specificity of serine hydroxymethyltransferase (SHMT) activities was tested in extracts of several methanogens using tetrahydromethanopterin (H4MPt) from Methanobacterium thermoautotrophicum Marburg, tetrahydrosarcinapterin (H4SPt) from Methanosarcina barkeri, and tetrahydrofolate (H4folate) as the potential C1 carrier. In Methanosphaera stadtmanae and Methanococcus thermolithotrophicus, the activities were H4MPt dependent. In Methanospirillum hungatei GP1, Methanosaeta concilii, Methanolobus tindarius, and Methanosarcina barkeri Fusaro, the activities were strictly H4folate dependent. H4SPt was reactive with the SHMT of Methanosphaera stadtmanae but not with that of Methanosarcina barkeri. In both Methanosarcina barkeri and Methanospirillum hungatei, pyridoxal phosphate stimulated SHMT activity. The apparent Km values for H4folate and L-serine were 0.086 and 0.29 mM in Methanosarcina barkeri and 0.065 and 0.31 mM in Methanospirillum hungatei, respectively. Key words: tetrahydromethanopterin, tetrahydrofolate, Archaea, serine hydroxymethyltransferase, methanogen.

Properties of the particulate enzyme F420-reducing hydrogenase isolated from Methanospirillum hungatei

1987 ◽  
Vol 33 (10) ◽  
pp. 896-904 ◽  
Author(s):  
G. Dennis Sprott ◽  
Kathleen M. Shaw ◽  
Terry J. Beveridge
Keyword(s):  
Gel Filtration ◽  
Methanobacterium Thermoautotrophicum ◽  
Methanosarcina Barkeri ◽  
Nonheme Iron ◽  
Anaerobic Conditions ◽  
Hydrophobic Properties ◽  
Iron Protein ◽  
Methanospirillum Hungatei ◽  
Cytoplasmic Membranes ◽  
Spectrum Characteristic

F420-reducing hydrogenase was isolated from spheroplast lysates of Methanospirillum hungatei by sedimentation, followed by sucrose gradient centrifugations and gel filtration. These procedures resulted in a preparation free of methyl reductase and cytoplasmic membranes. The hydrogenase was a brown protein with an absorption spectrum characteristic for a nonheme iron protein. In electron micrographs it was a coin-shaped, multisubunit protein complex of 15.9 nm diameter with a central depression on one surface. On phenyl Sepharose chromatography the hydrogenase exhibited hydrophobic properties. The holoenzyme was about 720 kilodaltons (kDa), composed of 50.7 and 30.7 kDa subunits in a ratio of 1:3. Each enzyme particle contained 6 or 7 Ni2+ atoms. H2-dependent reduction of F420 activity was readily, but transiently, reactivated by anaerobic conditions following exposure of the enzyme to air. Mg2+ or Ca2+ were stimulatory, but added FAD was not required. Antibody raised against the purified hydrogenase of strain GP1 gave a negative reaction with extracts of nine other methanogens and a reaction of identity with strain JF1 and Methanosarcina barkeri MS. Direct comparisons with the hyrogenase from Methanobacterium thermoautotrophicum ΔH revealed striking differences in subunit composition and in the acidity of the holoenzyme.

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge

1987 ◽  
Vol 33 (6) ◽  
pp. 551-554 ◽  
Author(s):  
Ken F. Jarrell ◽  
Michelle Saulnier ◽  
Art Ley
Keyword(s):  
Heavy Metals ◽  
Fatty Acids ◽  
Heavy Metal ◽  
Sewage Sludge ◽  
Metal Toxicity ◽  
Heavy Metal Toxicity ◽  
Methanosarcina Barkeri ◽  
Methanospirillum Hungatei ◽  
Nickel Zinc ◽  
Pure Cultures

The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobacterium formicicum at pH 6.5 was studied. The latter three strains were resistant to > 60 g/L of the volatile fatty acids and to > 10 g/L of NH3 N. Methanospirillum hungatei was somewhat more sensitive with 50% inhibition of methanogenesis occurring at 4.2 g/L NH3 N, 27 g/L butyrate, and 41 g/L propionate. All strains were very sensitive to both copper (1–5 mg/L) and zinc (1–10 mg/L), but much more resistant to nickel. Zinc and copper concentrations 30 to 270 times higher were required to cause inhibition of Msp. hungatei incubated in sewage sludge compared with buffer, indicating a strong protective environment was afforded the methanogens against heavy metal toxicity in the sludge.

Protein composition of Methanococcus thermolithotrophicus flagella

1992 ◽  
Vol 38 (11) ◽  
pp. 1162-1166 ◽  
Author(s):  
Alla S. Kostyukova ◽  
Georgi M. Gongadze ◽  
Anna Ya. Obraztsova ◽  
Konstantin S. Laurinavichus ◽  
Oleg V. Fedorov
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Key Words ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Complete Removal ◽  
Methanococcus Thermolithotrophicus ◽  
Molecular Weights ◽  
Dodecyl Sulfate

Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of flagella from the thermophilic methanogen Methanococcus thermolithotrophicus indicated that they were composed of three major proteins, with molecular weights of 62 000,44 000, and 26 000, whereas all previously studied flagella of mesophilic methanogens consisted of two subunits. Proteins were isolated by preparative electrophoresis followed by complete removal of sodium dodecyl sulfate and their renaturation. It was shown that at least two of the proteins contain a thermostable domain whose complete denaturation proceeds only upon prolonged boiling in the presence of sodium dodecyl sulfate. Key words: flagellin, thermostability, archaebacteria, Methanococcus thermolithotrophicus.

Methanogenesis in the absence of intracytoplasmic membranes

1984 ◽  
Vol 30 (5) ◽  
pp. 594-604 ◽  
Author(s):  
G. D. Sprott ◽  
L. C. Sowden ◽  
J. R. Colvin ◽  
K. F. Jarrell ◽  
T. J. Beveridge
Keyword(s):  
Membrane System ◽  
Accurate Method ◽  
Methanobacterium Thermoautotrophicum ◽  
Methane Formation ◽  
Bacterial Cells ◽  
Optimal Temperature ◽  
Intracytoplasmic Membrane ◽  
Methanospirillum Hungatei ◽  
Large Numbers ◽  
Intracytoplasmic Membranes

The frequency of intracytoplasmic membranes in several methanogens grown on H2–CO2 varied with the conditions of growth and varied from one strain to another. Methanobacterium thermoautotrophicum often generated large numbers of intracytoplasmic membranes, while Methanospirillum hungatei produced these membranes only rarely. Conditions allowing for rapid growth, including optimal temperature and high agitation rates, increased the production of intracytoplasmic membranes. These membranes consisted mainly of vesicles composed of one or several membrane layers, often positioned in the central region of the cytoplasm. Several mesophilic methanogens could be grown such that intracytoplasmic membranes were rarely or never observed in thin section or in replicas of cross-fractures from frozen cells. Since high rates of methane synthesis still occurred in these cultures, it follows that the intracytoplasmic membrane system is not a necessary organelle for methane formation in these strains. Negative staining for electron microscopy is not an accurate method to visualize intracytoplasmic membranes in these bacterial cells.

scholarly journals Similarity between serine hydroxymethyltransferase and other pyridoxal phosphate-dependent enzymes

FEBS Letters ◽  
1993 ◽  
Vol 331 (1-2) ◽  
pp. 145-149 ◽  
Author(s):  
Stefano Pascarella ◽  
Verne Schirch ◽  
Francesco Bossa
Keyword(s):  
Pyridoxal Phosphate ◽  
Serine Hydroxymethyltransferase

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transminase enzymes

1991 ◽  
Vol 332 (1263) ◽  
pp. 131-139 ◽  
Keyword(s):  
Active Site ◽  
Pyridoxal Phosphate ◽  
Isotope Effects ◽  
Serine Hydroxymethyltransferase ◽  
Side Chain ◽  
X Ray ◽  
Kinetic Isotope ◽  
Lysine Residues ◽  
Nitrogen Nucleophiles ◽  
Active Site Histidine

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C α and C-4'positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular p K a of the monoprotic base. Thus the ε-ammonium group of the internal aldimine-forming lysine residue operates at C-4'- si -face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C α from the 4'- si -face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldim ination processes and as a binding group for the α-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. — S— X— H — K — ) with that for aspartate aminotransferase (e.g. — S— X — A— K— ) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data. A similar model for serine hydroxymethyltransferase suggested that previous reports of stereochemical infidelity with decarboxylation substrates were incorrect. A series of careful experiments confirmed this. Hence, no actual examples of non-stereospecific α-amino acid decarboxylation by pyridoxal enzymes exist.

Oxidation–reduction of methanol, formaldehyde, serine, and formate in Methanosphaera stadtmanae using 14C short- and long-term labelling

1995 ◽  
Vol 41 (11) ◽  
pp. 1048-1053 ◽  
Author(s):  
Z. Lin ◽  
R. Sparling
Keyword(s):  
Dehydrogenase Activity ◽  
Formate Dehydrogenase ◽  
Serine Hydroxymethyltransferase ◽  
Whole Cells ◽  
Cytoplasmic Proteins ◽  
Oxidation Reduction ◽  
Methanosphaera Stadtmanae ◽  
Formaldehyde Oxidation ◽  
Short And Long Term

Methanosphaera stadtmanae derives its energy from the reduction by H2 of CH3OH, but not CO2, indicating there is a block in the CO2 methanogenesis pathway. Both 14CH4 and 14CO2 production were detected in whole cells using [14C]formaldehyde or [14C]serine as substrate. 14CO2 was also observed from [14C]formate in both whole cells and cofactor-depleted cell-free extracts, and NADP-dependent formate dehydrogenase activity was detected. Both formate and serine blocked the formation of 14CO2 from formaldehyde in whole cells. The results confirmed that enzymes involved in the reduction of carbon from the level of methylene-tetrahydromethanopterin in a common methanogenic pathway and a tetrahydromethanopterin-dependent serine hydroxymethyltransferase were present in this organism. However, the production of 14CH4 could not be observed from [14C]formate or 14CO2 plus H2. [14C]Formate was incorporated specifically into histidine and RNA. [14C]Methanol was also found to label rRNA and cytoplasmic proteins, especially corrinoid proteins.Key words: methanogenesis, formate dehydrogenase, formaldehyde oxidation, C1 intermediates.

Sign in / Sign up

Export Citation Format

Share Document