Purification and partial characterization of a putative thymidylate synthase from Methanobacterium thermoautotrophicum.

A protein catalyzing the tritium exchange of [5-3H]deoxyuridine monophosphate ([5-3H]dUMP) for solvent protons and the dehalogenation of 5-bromo-deoxyuridine monophosphate (Br-dUMP) has been isolated from the methanogenic archaea Methanobacterium thermoautotrophicum. These two activities are well-established side reactions of thymidylate synthase and do not require cofactors. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the purified enzyme showed a single band with a molecular mass of 27 kDa. The suggested molecular mass of the native protein calculated from sedimentation equilibrium experiments was 33.5 kDa, indicating that the enzyme is a monomer. The pH optima were 9.0 and 7.0 for the exchange reaction and the dehalogenation, respectively. The effects of temperature, salt, reducing agent and inhibitors were determined. The apparent Km for the tritium exchange from [5-3H]dUMP was 7 microM and for the dehalogenation of Br-dUMP was 14 microM. However, thus far, the conditions for dTMP synthesis from dUMP have not yet been established. Incubation of the enzyme with dUMP, tetrahydromethanopterin, a folate analog present in methanogens, and formaldehyde did not yield dTMP. The first 30 amino acids of the amino terminus have been sequenced. However, there is no similarity with any of the thymidylate synthases. Surprisingly, the protein from M. thermoautotrophicum appears to be related to chitin synthases from several organisms.

Dehalogenation of trichlorofluoromethane (CFC-11) by Methanosarcina barkeri.

Methanobacterium barkeri was found to catalyze the reductive dehalogenation of trichlorofluoromethane (CFC-11), also known as FREON 11. Products detected were CHFCl2, CH2FCl, CO and fluoride.

Reductive formation of carbon monoxide from CCl4 and FREONs 11, 12, and 13 catalyzed by corrinoids.

In an earlier publication, we reported that corrinoids catalyze the sequential reduction of CCl4 to CHCl3, CH2Cl2, CH3Cl, and CH4 with titanium(III) citrate as electron donor [Krone, U. E., Thauer, R. K., & Hogenkamp, H. P. C. (1989) Biochemistry 28, 4908-4914]. However, the recovery of these products was less than 50%, indicating that other products were formed. We now report that, under the same experimental conditions, CCl4 is also converted to carbon monoxide. These studies were extended to include FREONs 11, 12, 13, and 14. Corrinoids were found to catalyze the reduction of CFCl3, CF2Cl2, and CF3Cl to CO and, in the case of CFCl3, to a lesser extent, to formate. CF4 was not reduced. The rate of CO and formate formation paralleled that of fluoride release. Both rates decreased in the series CFCl3, CF2Cl2, CCl4, and CF3Cl. The reduction of CFCl3 gave, in addition to CO and formate, CHFCl2, CH2FCl, CH3F, C2F2Cl2, and C2F2Cl4. The product pattern indicates that the corrinoid-mediated reduction of halogenated C1-hydrocarbons involves the intermediacy of dihalocarbenes, which may be a reason why these compounds are highly toxic for anaerobic bacteria.

Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated C1 hydrocarbons in methanogenic bacteria.

Corrinoids, such as aquocobalamin, methylcobalamin, and (cyanoaquo)cobinamide, catalyze the reductive dehalogenation of CCl4 with titanium(III) citrate as the electron donor [Krone et al. (1989) Biochemistry 28, 4908-4914]. We report here that this reaction is also effectively mediated by the nickel-containing porphinoid, coenzyme F430, found in methanogenic bacteria. Chloroform, methylene chloride, methyl chloride, and methane were detected as intermediates and products. Ethane was formed in trace amounts, and several as yet unidentified nonvolatile compounds were also generated. The rate of dehalogenation decreased in the series of CCl4, CHCl3, and CH2Cl2. With coenzyme F430 as the catalyst, the reduction of CH3Cl to CH4 proceeded more than 50 times faster than with aquocobalamin. Cell suspensions of Methanosarcina barkeri were found to catalyze the reductive dehalogenation of CCl4 with CO as the electron donor (E'0 = -0.524 V). Methylene chloride was the main end product. The kinetics of CHCl3 and CH2Cl2 formation from CCl4 were similar to those with coenzyme F430 or aquocobalamin as catalysts and titanium(III) citrate as the reductant.