James T. Drummond

Cobalamin-dependent methionine synthase and serine hydroxymethyltransferase: Targets for chemotherapeutic intervention?

Chemotherapeutic drugs targeted at folate-dependent reactions have typically been directed at a limited number of target enzymes: dihydrofolate reductase, thymidylate synthase, and GAR and AICAR transformylase. This review discusses two other potential targets for chemotherapeutic inhibition: cobalamin-dependent methionine synthase and serine hydroxymethyltransferase. Brief reviews of the catalytic properties of these two enzymes are presented, and possible strategies for chemotherapeutic intervention are discussed.

Crystallization and preliminary X-ray diffraction studies of the cobalamin-binding domain of methionine synthase from Escherichia coli☆

Crystals of a cobalamin-binding domain (M(r) = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5, starting from solutions of intact (M(r) = 133,000) cobalamin-dependent methionine synthase. The crystals are orthorhombic in space group P2(1)2(1)2(1), with cell dimensions a = 96.9 A, b = 55.4 A, c = 103.8 A. For two molecules per asymmetric unit, the calculated VM value is 2.45 A3/Da. A native data set has been collected to 3 A resolution.

Cobalamin-Dependent and Cobalamin-Independent Methionine Synthases in Escherichia coli: Two Solutions to the Same Chemical Problem

Two genes encoding proteins with methionine synthase activity are found in Escherichia coli. Both enzymes use methyltetrahydrofolate as a methyl donor to catalyze the conversion of homocysteine to methionine, as shown below:

The interaction of nitrous oxide with cobalamin-dependent methionine synthase

C{H_3} -{H_4}PteGl{u_n} + Homocysteine(RHS) -------->{H_4}PteGl{u_n} + Methionine(RSC{H_3})

Providing One-Carbon Units for Biological Methylations: Mechanistic Studies on Serine Hydroxymethyltransferase, Methylenetetrahydrofolate Reductase, and Methyltetrahydrofolate-Homocysteine Methyltransferase

The metH gene encodes a cobalamin-dependent methionine synthase (MetH). This protein is monomelic, with a deduced molecular weight of 136,055.1 It can use CH3-H4PteGlu1 as a substrate, and requires a reducing system and AdoMet for activation.2,3 The metE gene encodes a cobalamin-independent methionine synthase that requires methyltetrahydrofolate substrates with at least two glutamyl residues and is completely inactive with CH3-H4PteGlu1.4 This enzyme shows no requirement for reductive activation, but does require phosphate ion and magnesium for optimal rates of catalysis.5 It has a deduced molecular weight of 84,654,6 in good agreement with the value of 84,000 obtained by ultracentrifugation of the native enzyme.5 Both the metH 1,7,8 and metE 6,9genes have been sequenced, and their coding sequences show no detectable homologies. Thus these two proteins appear to have evolved independently to perform highly similar methyl transfers from N5 of methyltetrahydrofolate to the sulfur of homocysteine.

Assignment of Enzymatic Function to Specific Protein Regions of Cobalamin-Dependent Methionine Synthase from Escherichia coli

Mismatch recognition in human cells is mediated by two heterodimers, MutS alpha and MutS beta. MutS alpha appears to shoulder primary responsibility for mismatch correction during replication, based on its relative abundance and ability to recognize a broad spectrum of base-base and base-insertion mismatches. Because MutS alpha and MutS beta share a common component, MSH2, conditions that influence the expression or degradation of MSH3 or MSH6 can redistribute the profile of mismatch recognition and repair. MSH3 is linked by a shared promoter with DHFR, connecting two pathways with key roles in DNA metabolism. In a classic example of gene amplification, the DHFR (and MSH3) locus can become amplified to several hundred copies in the presence of methotrexate. Under these conditions, MutS beta forms at the expense of MutS alpha, and the mutation rate in these tumor cells rises more than 100-fold. The implications for cancer chemotherapy include a potential increase in mutability when tumors are treated with methotrexate, which could increase the frequency of subsequent mutations that influence the tumors drug sensitivity or aggressiveness. Because processing certain types of DNA damage by the mismatch repair pathway has also been implicated in tumor sensitivity to agents such as cisplatin, changes in expression at the DHFR/MSH3 locus may have further relevance to the outcome of multi-drug treatment regimens.

Characterization of nonradioactive assays for cobalamin-dependent and cobalamin-independent methionine synthase enzymes.

Prolonged treatment of patients with the commonly used anaesthetic, nitrous oxide, results in the development of the symptoms of acute cobalamin deficiency. Inactivation of cobalamin-dependent methionine synthase has been demonstrated under these conditions. We have previously demonstrated that methionine synthase can also be inactivated in vitro during turnover under an atmosphere of nitrous oxide [ 11. However, only catalytic amounts of the enzyme could be inactivated, because inactivation is a relatively rare event during turnover. We have now developed a method of redox cycling that permits inactivation of several pmoles of purified enzyme so that chemical events associated with this inactivation can be elucidated. The enzyme is poised in an electrochemical cell in the presence of methyl viologen and KC1 under an atmosphere of N20. Because we have previously measured the standard reduction potential of the enzyme-bound cob(II)alamin/cob(I)alamin couple [2], we can determine the concentration of cob(I)alamin present at equilibrium at the applied potential. We have been able to show that the enzyme loses activity in a first order fashion during redox cycling under N20, and that the rate of inactivation varies linearly with the concentration of cob(I)alamin present at the applied potential. Our results are simply interpreted as indicating that inactivation is associated with reduction of nitrous oxide by enzyme-bound cob(I)alamin. Further experiments to characterize the oxygen product derived from N20 and the inactive protein are currently in progress.