Evidence that the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate is a product of the methylreductase reaction in Methanobacterium

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.     

Coupling of methyl coenzyme M reduction with carbon dioxide activation in extracts of Methanobacterium thermoautotrophicum

The stimulation of carbon dioxide reduction to methane by addition of 2-(methylthio)ethanesulfonate (CH3-S-CoM) to cell extracts of Methanobacterium thermoautotrophicum was investigated. Similar stimulation of CO2 reduction by CH3-S-CoM was found for cell extracts of Methanobacterium bryantii and Methanospirillum hungatei. The CH3-S-CoM requirement could be met by the methanogenic precursors formaldehyde, serine, or pyruvate, or by 2-(ethylthio)ethanesulfonate (CH3CH2-S-CoM), but not by other coenzyme M derivatives. Efficient reduction of CO2 to CH4 was favored by low concentrations of CH3-S-CoM and high concentrations of CO2. Sulfhydryl compounds were identified as effective inhibitors of CO2 reduction. Both an allosteric model and a free-radical model for the mechanism of CO2 activation and reduction are discussed.     

Transport of coenzyme M (2-mercaptoethanesulfonic acid) and methylcoenzyme M [(2-methylthio)ethanesulfonic acid] in Methanococcus voltae: identification of specific and general uptake systems.

A transport system for coenzyme M (2-mercaptoethanesulfonic acid [HS-CoM]) and methylcoenzyme M [(2-(methylthio)ethanesulfonic acid (CH3-S-CoM)] in Methanococcus voltae required energy, showed saturation kinetics, and concentrated both forms of coenzyme M against a concentration gradient. Transport required hydrogen and carbon dioxide for maximal uptake. CH3-S-CoM uptake was inhibited by N-ethylmaleimide and monensin. Both HS-CoM and CH3-S-CoM uptake showed sodium dependence. In wild-type M. voltae, HS-CoM uptake was concentration dependent, with a Vmax of 960 pmol/min per mg of protein and an apparent Km of 61 microM. Uptake of CH3-S-CoM showed a Vmax of 88 pmol/min per mg of protein and a Km of 53 microM. A mutant of M. voltae resistant to the coenzyme M analog 2-bromoethanesulfonic acid (BES) showed no uptake of CH3-S-CoM but accumulated HS-CoM at the wild-type rate. While the higher-affinity uptake system was specific for HS-CoM, the lower-affinity system mediated uptake of HS-CoM, CH3-S-CoM, and BES. Analysis of the intracellular coenzyme M pools in metabolizing cells showed an intracellular HS-CoM concentration of 14.8 mM and CH3-S-CoM concentration of 0.21 mM.     

An unusual thiol-driven fumarate reductase in Methanobacterium with the production of the heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine-O3-phosphate

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.     

The final step in methane formation. Investigations with highly purified methyl-CoM reductase (component C) from Methanobacterium thermoautotrophicum (strain Marburg)

Methyl-coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3-S-CoM) with N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP = component B) to CH4. The mixed disulfide of H-S-CoM and H-S-HTP (CoM-S-S-HTP) was the other major product formed. The specific activity was up to 75 nmol min-1 mg protein-1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3-S-CoM reduction to CH4 with H-S-HTP increased to 0.5-2 mumol min-1 mg protein-1. Under these conditions the CoM-S-S-HTP formed from CH3-S-CoM and H-S-HTP was completely reduced to H-S-CoM and H-S-HTP. Methyl-CoM reductase was specific for H-S-HTP as electron donor. Neither N-6-mercaptohexanoylthreonine phosphate (H-S-HxoTP) nor N-8-mercaptooctanoylthreonine phosphate (H-S-OcoTP) nor any other thiol compound could substitute for H-S-HTP. On the contrary, H-S-HxoTP (apparent Ki = 0.1 microM) and H-S-OcoTP (apparent Ki = 15 microM) were found to be effective inhibitors of methyl-CoM reductase, inhibition being non-competitive with CH3-S-CoM and competitive with H-S-HTP.     

Methanofuran (carbon dioxide reduction factor), a formyl carrier in methane production from carbon dioxide in Methanobacterium

Methanofuran (carbon dioxide reduction factor) became labeled when incubated in cell extracts of Methanobacterium under hydrogen and 14CO2 in the absence of methanopterin. Proton NMR spectroscopy revealed that a formyl group was bound to the primary amine of methanofuran. [14C]Formylmethanofuran was enzymically converted to 14CH4 in the presence of CH3-S-CoM [2-(methylthio)ethanesulfonic acid], hydrogen, and methanopterin, establishing the formyl moiety as an intermediate in methanogenesis. In the absence of methanopterin, a substantial portion of the formyl label was oxidized to 14CO2 rather than reduced to 14CH4, consistent with a model in which the C1 intermediate is first bound to methanofuran and then to methanopterin, during its reduction. When CH3-S-CoM was replaced by HS-CoM (2-mercaptoethanesulfonic acid), most of the formyl label was oxidized to 14CO2, indicating that methyl group reduction by the CH3-S-CoM methylreductase is required for the conversion of formylmethanofuran to methane.     

Detection of a glycosylated subunit in human serum ferritin.

Chemical reaction of coenzyme M, sodium 2-mercaptoethanesulphonate (HS-CoM, Na+), and formaldehyde formed sodium 2-(hydroxymethylthio)ethanesulphonate (HOCH2-S-CoM), whereas reaction with the ammonium salt of HS-CoM yielded iminobis-[2-(methylthio)ethanesulphonate], monoammonium salt [NH = (CH2 - S - CoM)2]. In water, NH = (CH2 - S - CoM)2 decomposed to 2-(aminomethylthio)ethanesulphonate (NH2CH2 - S - CoM) and HOCH2-S-CoM. NH-2-CH2 - CoM was degraded further to form more HOCH2-S-CoM. The structures of these coenzyme M derivatives were confirmed by i.r. and n.m.r. spectroscopy and by elemental analysis. When added to cell extracts of Methanobacterium thermoautotrophicum, methane was formed from either HOCH2 - S - CoM or NH = (CH2 - S - CoM)2 at rates comparable with the rate of methane formation from the methanogenic precursor 2-(methylthio)-ethanesulphonate (CH3 - S - CoM). Formaldehyde was reduced to methane at similar rates. In addition, certain hemimercaptals, including thiazolidine and thiazolidine-4-carboxylate, were reduced, although at slower rates. The reduction of formaldehyde, thiazolidine, or thiazolidine-4-carboxylate required catalytic amounts of HS-CoM. ATP was required by cells extracts for reduction of each of these methane precursors.     

Preparation of coenzyme M analogues and their activity in the methyl coenzyme M reductase system of Methanobacterium thermoautotrophicum.

A number of 2-(methylthio)ethanesulfonate (methyl-coenzyme M) analogues were synthesized and investigated as substrates for methyl-coenzyme M reductase, an enzyme system found in extracts of Methanobacterterium thermoautotrophicum. Replacement of the methyl moiety by an ethyl group yielded an analogue which served as a precursor for ethane formation. Propyl-coenzyme M, however, was not converted to propane. Analogues which contained additional methylene carbons such as 3-(methylthio)propanesulfonate or 4-(methylthio)butanesulfonate or analogues modified at the sulfide or sulfonate position, N-methyltaurine and 2-(methylthio)ethanol, were inactive. These analogues, in addition to a number of commercially available compounds, also were tested for their ability to inhibit the reduction of methyl-coenzyme M to methane. Bromoethanesulfonate and chloroethanesulfonate proved to be potent inhibitors of the reductase, resulting in 50% inhibition at 7.9 X 10(6) M and 7.5 X 10(5) M. Analogues to coenzyme M which contained modifications to other regions were evaluated also and found to be weak inhibitors of methane biosynthesis.     

Characterization of bromoethanesulfonate-resistant mutants of Methanococcus voltae: evidence of a coenzyme M transport system.

Mutants of Methanococcus voltae were isolated that were resistant to the coenzyme M (CoM; 2-mercaptoethanesulfonic acid) analog 2-bromoethanesulfonic acid (BES). The mutants displayed a reduced ability to accumulate [35S]BES relative to the sensitive parental strain. BES inhibited methane production from CH3-S-CoM in cell extracts prepared from wild-type sensitive or resistant strains. BES uptake required the presence of both CO2 and H2 and was inhibited by N-ethylmaleimide and several reagents that are known to disrupt energy metabolism. The mutants showed normal uptake of isoleucine and were not cross-resistant to either azaserine or 5-methyltryptophan and, thus, were neither defective in general energy-dependent substrate transport nor envelope permeability. Both HS-CoM and CH3-S-CoM prevented the uptake of BES and protected cells from inhibition by it. We propose that M. voltae has an energy-dependent, carrier-mediated uptake system for HS-CoM and CH3-S-CoM which can also mediate uptake of BES.     

The interaction of adeninylalkylcobalamins with ribonucleotide reductase.

Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.     

The interaction of adeninylalkycobalamins with ribonucleotide reductase

Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.     

Coordination and geometry of the nickel atom in active methyl-coenzyme M reductase from Methanothermobacter marburgensis as detected by X-ray absorption spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH(3)-S-CoM) to methane. The enzyme contains as a prosthetic group the nickel porphinoid F(430) which in the active enzyme is in the EPR-detectable Ni(I) oxidation state. Crystal structures of several inactive Ni(II) forms of the enzyme but not of the active Ni(I) form have been reported. To obtain structural information on the active enzyme-substrate complex we have now acquired X-ray absorption spectra of active MCR in the presence of either CH(3)-S-CoM or the substrate analog coenzyme M (HS-CoM). For both MCR complexes the results are indicative of the presence of a five-coordinate Ni(I), the five ligands assigned as four nitrogen ligands from F(430) and one oxygen ligand. Analysis of the spectra did not require the presence of a sulfur ligand indicating that CH(3)-S-CoM and HS-CoM were not coordinated via their sulfur atom to nickel in detectable amounts. As a control, X-ray absorption spectra were evaluated of three enzymatically inactive MCR forms, MCR-silent, MCR-ox1-silent and MCR-ox1, in which the nickel is known to be six-coordinate. Comparison of the edge position of the X-ray absorption spectra revealed that the Ni(I) in the active enzyme is more reduced than the Ni in the two EPR-silent Ni(II) states. Surprisingly, the edge position of the EPR-active MCR-ox1 state was found to be the same as that of the two silent states indicating similar electron density on the nickel.     

Enzymic reactions of phosphate analogs.

A variety of phosphonates (XPO32-; X = H-, CH3-, CL3C-, CH3CH2-, and phenyl-) as well as methylarsonate have been shown to be suitable phosphate analogs for the reactions catalyzed by yeast glyceraldehyde-3-phosphate dehydrogenase and calf spleen purine nucleoside phosphorylase. The reactivity of the phosphate analogs with these two enzymes is independent of the pKa of the analog.     

Identification of methyl coenzyme M as an intermediate in methanogenesis from acetate in Methanosarcina spp

The transfer of the methyl group of acetate to coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) during the metabolism of acetate to methane was investigated in cultures of Methanosarcina strain TM-1. The organism metabolized CD3COO- to 83% CD3H and 17% CD2H2 and produced no CDH3 or CH4. The isotopic composition of coenzyme M in cells grown on CD3COO- was analyzed with a novel gas chromatography-mass spectrometry technique. The cells contained CD3-D-CoM and CD2H-S-CoM) in a proportion similar to that of CD3H to CD2H2. These results, in conjunction with a report (J.K. Nelson and J.G. Ferry, J. Bacteriol. 160:526-532, 1984) that extracts of acetate-grown strain TM-1 contain high levels of CH3-S-CoM methylreductase, indicate that CH3-S-CoM is an intermediate in the metabolism of acetate to methane in this organism.     

Substrate analogues as mechanistic probes of methyl-S-coenzyme M reductase

Methyl-S-coenzyme M reductase catalyzes the ultimate methane-yielding reaction in methanogenic bacteria, the reductive cleavage of the terminal carbon-sulfur bond of 2-(methylthio)ethanesulfonic acid. This protein has previously been shown to contain 2 equiv of a tightly bound nickel corphinoid cofactor, denoted cofactor F430, that may play a role in catalysis. Prior to this study, only one substrate analogue, ethyl-S-coenzyme M, had been demonstrated to be processed to a product by anaerobic cell extracts from Methanobacterium thermoautotrophicum strain delta H. In this investigation, we have synthesized three additional substrate analogues that serve as substrates as well as five previously unknown inhibitors. Steady-state kinetic techniques were developed in order to assess relative rates of processing for these substrates and inhibitors by use of anaerobic cell extracts from M. thermoautotrophicum. With this assay system, a KM of 0.1 mM and a kcat of 17 min-1 were determined for methyl-S-coenzyme M as substrate. Methyl-seleno-coenzyme M was converted to methane with a kcat threefold higher than that of methyl-S-coenzyme M, but kcat/KM was unchanged. The carbon-oxygen bond of 2-methoxyethanesulfonic acid was not cleaved to yield methane, but this analogue acted as an inhibitor with a K1 of 8.3 mM. Methyl reductase catalyzed reductive cleavage of difluoromethyl-S-coenzyme M to yield difluoromethane as the sole product, but trifluoromethyl-S-coenzyme M and trifluoromethyl-seleno-coenzyme M were inhibitors and not substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the carbohydrate moiety bound to N-7-mercaptoheptanoyl-O-threonine phosphate

Abstract Component B ( N -7-mercaptoheptanoyl-threonine- O -3-phosphate) (HS-HTP) which is an absolute requirement in the methylcoenzyme M methylreductase reaction was found to be part of a complex UDP-disaccharide when isolated carefully from cell-free supernant of Methanobacterium thermoautotrophicum . The site of attachment of HS-HTP to the UDP-disaccharide was through a carboxylic-phosphoric anhydride linkage of the C-6 mannosaminuronic acid to the phosphate group in HS-HTP. This bond is quite labile and this may account for the fact that the intact molecule, called methyl reducing factor (MRF) was not isolated previously. The structure of MRF was determined by combined fast atom bombardment mass spectrometry and ¹H-, ¹³C-, and ³¹P-NMR spectroscopy and assigned as: uridine 5'-[ N -7-mercaptoheptanoyl- O -3-phosphothreonine(2-acetamido-2-deoxy- β -mannopyranuronosyl)acid anhydride]-(1 → 4)- O -2-acetamido-2-deoxy α -glucopyranosyl diphosphate.     

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (K _i =150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme m, 2-(methylthio)ethanesulfonate - HS-HTP 7-mercaptoheptanoyl-l-threonine phosphate - CoM-SS-HTP the heterodisulfide of HS-CoM and HS-HTP - BCFE bolled cell-free extract - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - PEP phosphoenolpyruvate - 2,3-DPG 2,3-diphosphoglycerate - cDPG cyclic 2,3-diphosphoglycerate - 3-PG 3-phosphoglycerate - 2-PG 2-phosphoglycerate     

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs

A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.     

Relationship between structure and P-glycoprotein inhibitory activity of dimeric peptides related to the Dmt-Tic pharmacophore

To develop novel inhibitors of P-glycoprotein (P-gp), dimeric peptides related to an opioid peptide containing the Dmt-Tic pharmacophore were synthesized and their P-gp inhibitory activities were analyzed. Of the 30 analogs synthesized, N(α),N(ε)-[(CH(3))(2)Mle-Tic](2)Lys-NH(2) and its D-Lys analog were found to exhibit potent P-gp inhibitory activity, twice that of verapamil, in doxorubicin-resistant K562 cells. Structure-activity studies indicated that the correct hydrophobicity and spacer length between two aromatic rings are important structural elements in this series of analogs for inhibition of P-gp.     

Biochemistry of methanogenesis.

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH3-S-CoM) to CH4. Seminal studies on the CO2-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO2 to the methyl level (CH3-S-CoM) with electrons from H2 or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH3-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO2 by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH3-S-CoM; electrons for reduction of CH3-S-CoM to CH4 are provided by the oxidation of methyl groups to CO2.