Abstract
Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.
Highlights
Evidence obtained by I3C NMR spectroscopy indi- labile coenzyme involved in the disproportionation of HCHO cates that tetrahydromethanopterin (H4MPT) serves under a nitrogen atmosphere and has been isolated in a highly as a carbon carrier for C, units at the methine, meth- purified form from deproteinated extracts of Methanobacterylene, and methyl levels of oxidation
All three deriv- ium thermoautotrophicumstrain AH [15].The chemical strucatives of H4MPT served as substrates for methano- ture of H4MPT has been recently elucidated [26]. In this genesis by cell extracts under a hydrogen atmosphere; communication, we present results obtained from UV specin each instance, methaneevolved at a rate comparable troscopy, 13C and 'H NMR spectroscopy, and HRFABMS, to that obtained when 2-(methy1thio)ethanesulfonic which indicate that H,MPT is a carbon carrier in methanoacid was used as the substrate
HCHO and H,MPT react chemically to form an adduct documented by HRFABMS to be methylene-H4MPT
Summary
Dehyde with H4MPTwas methylene-H4MPT,with the molecular formula C31H45N6016PI.3C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methyleneH4MPT was bound to two nitrogen atoms. Riboflavin 5'-phosphate; HRFABMS, high resolution fast atom bom- To follow the effect of H2 on the reaction, a known amount of it bardment mass spectrometry; HIMPT, tetrahydromethanopterin; was injected into each reaction vial after the atmosphere in the CH&CoM, 2-(methythio)ethanesulfonic acid; Pipes, piperazine- headspace had been exchanged with NP.Before adding H2, an equal. The sample was brought into an anaerobic chamber, transferred into a Bausch and Lomb reaction cell, and removed from the chamber; the atmosphere in the headspace of the reaction cell was exchanged for Nz. After preincubation a t 60 "C for 10 min, the reaction was started by the addition of 70% ammonium sulfate-treated extract (6 pg of protein). Methenyl-H4MPT+ was obtained by enzymic oxidation of methylene-H4MPT (2 pmol) by Sephadex G-25-treated cell-free extract (1mg of protein) as described above. Protein concentration was determined by the turbidity of the sample in 20% trichloroacetic acid [20]
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