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.
Krone
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Department of Biochemistry, University of Minnesota, Minneapolis 55455.
McFarlan
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SC
Hogenkamp
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HP
D000595:Amino Acid Sequence; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D017014:Methanobacterium / Q000201:enzymology*; D008969:Molecular Sequence Data; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000302:isolation & purification*
D003856:Deoxyuracil Nucleotides; D013938:Thymidine Monophosphate; D013940:Thymidylate Synthase