7-mercaptoheptanoylthreonine Phosphate Research Articles

A novel biosynthesis of medium chain length α-ketodicarboxylic acids in methanogenic archaebacteria

Gas chromatographic-mass spectrometric analysis on the distribution of α-ketodicarboxylic acids in various bacteria determined that α-ketoglutarate and α-ketoadipate are widely distributed in all the bacteria examined, whereas α-ketopimelate and α-ketosuberate are found only in the methanogenic archaebacteria. Labeling experiments with stable isotopes indicated that each of these acids arises from α-ketoglutarate by repeated α-ketoacid chain elongation. The final product in this series of reactions, α-ketosuberate, serves in the methanogenic bacteria as the biosynthetic precursor to the 7-mercaptoheptanoic acid portion of 7-mercaptoheptanoylthreonine phosphate, a methanogenic coenzyme.

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH4 and CO2 formation from acetyl-CoA with specific activities of 50 nmol·min-1·mg protein-1. CH4 formation was found to be dependent on tetrahydromethanopterin (H4MPT) (apparent K M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F420 (F420). Methyl-H4MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H4MPT, and CH3-S-CoM as intermediates. The disproportionation of formaldehyde to CO2 and CH4 was also studied. This reaction was shown to be dependent on H4MPT, MFR, F420, H-S-CoM, and H-S-HTP.

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from [formula omitted][formula omitted

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5′-( O-2-acetamido-2-deoxy-β-mannopyranuronosyl acid-(1→4)-2-acetamido-2-deoxy-α-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

Biosynthesis of the 7-mercaptoheptanoic acid subunit of component B [(7-mercaptoheptanoyl)threonine phosphate] of methanogenic bacteria

Deuterium- and {sup 13}C-labeled precursors were used to establish the pathway for the biosynthesis of the 7-mercaptoheptanoic acid moiety of component B in methanogenic bacteria. The extent and position of the label incorporated into 7-mercaptoheptanoic acid were measured from the molecular and fragment ions in the mass spectrum of the methyl ester methylthiol derivative of the 7-mercaptoheptanoic acid. Deuterium from (2,2,2-{sup 2}H{sub 3})acetate was found to be incorporated into four separate positions of 7-mercaptoheptanoic acid. One deuterium was equally distributed between the C-2 and the C-3 of the 7-mercaptoheptanoic acid, and the remaining three were at carbons 4-6. The extent of incorporation of the C-2 and C-3 positions was the same as that observed for the incorporation of (2,2,2-{sup 2}H{sub 3})acetate into the {alpha}-ketoglutarate produced by the cells. (1,2-{sup 13}C{sub 2})Acetate was incorporated into four separate sites of the 7-mercaptoheptanoic acid molecule. On the basis of this and additional information, it is concluded that 7-mercaptoheptanoic acid is biosynthesized from {alpha}-ketosuberate, which arises from {alpha}-ketoglutarate by repeated {alpha}-keto acid chain elongation. The mechanism for the conversion of {alpha}-ketosuberate to a thiol appears to be analogous to that for the conversion of sulfopyruvate to coenzyme M (2-mercaptoethanesulfonic acid).

Reductive activation of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H.

When titanium(III) citrate was used as electron donor for the reduction of methyl coenzyme M by the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H, component A1 was no longer required. The simpler system thus obtained required components A2, A3, and C as well as catalytic amounts of ATP, vitamin B12, and the disulfide of 7-mercaptoheptanoylthreonine phosphate in addition to titanium(III) citrate. This three component enzyme system also could produce CH4 when stoichiometric amounts of 7-mercaptoheptanoylthreonine phosphate were used as a source of electrons under an H2 atmosphere. When 7-mercaptoheptanoylthreonine phosphate or H2 was used alone no CH4 was produced, indicating a dual requirement for reducing equivalents: one to activate the methylreductase system and the other to reduce methyl coenzyme M. This is the first evidence that the activation of methyl coenzyme M methylreductase is a reductive process.

Involvement of a corrinoid enzyme in methanogenesis from acetate inMethanosarcina barkeri

Abstract Extracts of acetate-grown Methanosarcina barkeri strain Fusaro formed methane from acetate plus ATP and form acetyl phosphate under H 2 . Coenzyme A (CoA) is stimulatory. Inhibitors of methanogenesis are cyanide, propyliodide and bromoethanesulfonic acid. In cofactor-free extracts methanogenic activity from acetate was restored by addition of ATP, CoA, coenzyme M and 7-mercaptoheptanoylthreonine phosphate. An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate.

Methanobacterium thermoautotrophicum contains a soluble enzyme system that specifically catalyzes the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate with H 2

Cell extracts of Methanobacterium thermoautotrophicum (strain Marburg) were found to catalyze the reduction of the heterodisulfide of coenzyme M (CoM-S-H) and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with H 2. All the activity was associated with the soluble cell fraction (160 000 × g supernatant). The enzyme system was purified sevenfold by anion-exchange chromatography. The partially purified system had a specific activity of 100 nmol CoM-S-S-HTP reduced per min and mg protein and exhibited an apparent K m for CoM-S-S-HTP of below 0.1 mM. The homodisulfides of CoM-S-H, of H-S-HTP, of cysteine, and of glutathione were not reduced. NADPH and NADH could not substitute for H 2 as electron donor.

Physiological importance of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate in the reduction of carbon dioxide to methane in Methanobacterium.

The heterodisulfide of the two coenzymes 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) and N-(7-mercaptoheptanoyl)threonine O3-phosphate (HS-HTP) increased the rate of CO2 reduction to methane by cell extracts 42-fold. The stimulation resulted from activation of the initial step of methanogenesis, the production of formylmethanofuran from methanofuran and CO2. These results establish a role for this heterodisulfide (CoM-S-S-HTP) in the reduction of CO2 to formylmethanofuran. Evidence indicates that CoM-S-S-HTP is the labile intermediate that accounts for the coupling of the reduction of 2-(methylthio)ethanesulfonic acid by the methylreductase to formylmethanofuran biosynthesis, the "RPG effect." The heterodisulfide was found to be labile in cell extracts due to enzyme-catalyzed reduction and possibly thioldisulfide exchange.

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 (CH 3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain ΔH, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH 3-S-CoM → CH 4 + CoM-S-S-HTP. CH 4 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 CH 3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.

Structure of purified cytoplasmic cofactor from methanobacterium thermoautotrophicum

The reduction of methylcoenzyme M to methane is known to require a heat stable and oxygen sensitive cofactor. Recently it has been shown that the active site of this cofactor is 7-mercaptoheptanoylthreonine phosphate. The present study shows that in the complete structure of this cofactor 7-mercaptoheptanoylthreonine phosphate is linked by pyrophosphate to two N-acetyl-glucosamine residues and an unidentified terminal group R with m/z 214. By fast-atom-bombardment mass spectrometry the intact cofactor, isolated as the mixed disulfide with 2-mercaptoethanol, was shown to have a molecular weight of 1084.5. The pyrophosphate bond is quite labile and undergoes hydrolysis or prolonged storage. This lability of the pyrophosphate bond may explain why the intact cofactor has not been isolated until now.

The role of 7-mercaptoheptanoylthreonine phosphate in the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum

The structure of component B of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum was recently found to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP). Three potential roles for this cofactor were considered. First, a methyl thioether derivative of the cofactor was synthesized to investigate its possible role as a methyl donor. This derivative was found to be incapable of acting as a substrate for methanogenesis and proved inhibitory. Secondly, an adenylated form of the cofactor was considered as the potential active form of the coenzyme. This possibility was ruled out based upon collaborative observations with Ankel-Fuchs et al. (FEBS Lett., in press) that HS-HTP is required by the methylreductase system even when ATP is not. Finally, HS-HTP was found to act as a reductant in a partially-purified methylreductase preparation that was incubated under nitrogen. The rate of methane production from HS-HTP exceeded that from other thiols or hydrogen.

7-Mercaptoheptanoylthreonine phosphate functions as component B in ATP-independent methane formation from methyl-CoM with reduced cobalamin as electron donor

Purified methyl-CoM reductase of Methanobacterium thermoautotrophicum (strain Marburg) catalyzed the reduction of methyl-CoM to methane with reduced cobalamin, when either synthetic 7-mercaptoheptanoyl-threonine phosphate (HS-HTP) or naturally occurring component B was present. With both compounds the same maximal specific acitivity was obtained and ATP was neither required nor stimulatory. These findings indicate that HS-HTP functions as component B and do not support the idea that HS-HT is only active in an adenosine monophosphorylated form.

Biochemical aspects of methane formation in Methanobacterium thermoautotrophicum.

The biochemistry of methanogenesis is characterized by the involvement of unique coenzymes [1]. Consequently, investigation of the enzymology of the pathway by which carbon dioxide is reduced to methane has been severely constrained. Until recently, only a single substrate of the pathway was known, 2-(methylthio)ethanesulfonate (CH3-S-CoM).The methylreductase system which catalyzes the reduction of CH3-S-CoM to methane has been extensively studied, but here, too, lack of knowledge of the components involved has hampered progress. The enzyme system consists of at least four proteins and displays an absolute requirement for a heat-stable cofactor, component B [2]. We have recently established that component B is 7-mercaptoheptanoylthreonine phosphate (HS-HTP) [3]. This structure was proposed based on analysis of the authentic cofactor and was confirmed by its complete chemical synthesis

Synthesis of 7-mercaptoheptanoylthreonine phosphate and its activity in the methylcoenzyme M methylreductase system.

The structure of component B of the methylcoenzyme M methylreductase of Methanobacterium thermoautotrophicum was recently assigned as 7-mercaptoheptanoylthreonine phosphate (HS-HTP) (Noll, K. M., Rinehart, K. L., Jr., Tanner, R.S., and Wolfe, R.S. (1986) (Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242). We report here the chemical synthesis and biochemical activity of this compound. Thiourea and 7-bromoheptanoic acid were used to to synthesize 7,7'-dithiodiheptanoic acid. This disulfide was then condensed with DL-threonine phosphate using N-hydroxysuccinimide and dicyclohexylcarbodiimide. The product was reduced with dithiothreitol to give HS-HTP. It could be oxidized in air in the presence of 2-mercaptoethanol to give the compound as it was isolated from cell extracts. The resulting product was identical to the authentic compound by 1H NMR spectroscopy, mass spectrometry, and coelution using high performance liquid chromatography. The synthetic compound is active in the in vitro methanogenic assay at concentrations comparable to the authentic compound. This confirms the structure of component B as HS-HTP and provides a means to synthesize quantities sufficient for studies of the methylreductase system.

Component C of the methylcoenzyme M methylreductase system contains bound 7-mercaptoheptanoylthreonine phosphate (HS-HTP)

The structure of component B of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum was recently found to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP). The work described here demonstrates that this compound is found in two forms: enzyme-free and enzyme-bound. HS-HTP was found to be bound to component C of the methylcoenzyme M methylreductase system. The cofactor extracted from the protein by heat denaturation was found to comigrate with the mixed disulfide of HS-HTP and 2-mercaptoethanol by high-performance liquid chromatography, suggesting HS-HTP is not modified in the bound state.

Structure of component B (7-mercaptoheptanoylthreonine phosphate) of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum.

Component B, the heat-stable low-molecular-weight cofactor required for methane production by dialyzed cell-free extracts of Methanobacterium thermoautotrophicum, has been purified to homogeneity and its structure assigned. Results of low-resolution fast-atom-bombardment and field-desorption mass spectrometry indicated a molecular weight of 419, and high-resolution fast-atom-bombardment mass spectrometry agreed with the molecular formula C13H26NO8PS2. Evidence from fast-atom-bombardment and field-desorption mass spectrometry and 360-MHz 1H NMR in deuterium oxide argued that the compound was isolated as a mixed disulfide with 2-mercaptoethanol; so the proposed elemental formula of the free acid, free thiol would be C11H22NO7PS (molecular weight, 343). The proposed structure for an active form of the coenzyme is 7-mercaptoheptanoylthreonine phosphate.