Folate-Dependent Enzymes | Cobalamin-Dependent Enzymes | Activation of Homocysteine
Using a variety of methods and tools, Dr. Matthews studies the mechanisms of folate-dependent enzymes and their regulation. She has succeeded in purifying methylenetetrahydrofolate reductase (MTHFR) to homogeneity in 1982 and later obtained the peptide sequence for about 40% of the enzyme. Dr. Matthews has collaborated with Professor Rima Rozen at McGill University to clone the human cDNA specifying methylenetetrahydrofolate reductase and they were subsequently able to identify a polymorphism in the human enzyme, C677T, which results in the substitution of Alanine for Valine.
This common mutation in MTHFR made it a candidate for a genetic risk factor for vascular disease. The mutation is associated with mild elevations in plasma homocysteine, especially in patients with a low folate status, and is thought to be a risk factor for cardiovascular disease and for the development of neural tube defects in the fetus. MTHFR or other folate derivatives with affinity for the enzyme, protect the mutant enzyme against loss of flavin and activity.
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Dr. Matthews and her colleagues have also studied cobalamin-dependent (MetH) and cobalamin-independent (MetE) methionine synthase. These two enzymes are unrelated in primary sequence, but catalyze highly similar reactions. The genes specifying each enzyme were sequenced by the Matthews laboratory. MetH was shown to be a highly modular protein, with domains responsible for homocysteine binding and activation, methylenetetrahydrofolate binding and activation, B12 binding, and reductive activation of the enzyme. In collaboration with U-M structural biologist Martha Ludwig, x-ray structures of the B12-binding region and the activation domain have been determined. The most striking feature of the structure of the cobalamin-binding region is the conformational change associated with binding of the methylcobalamin cofactor.
Methionine synthase, and its substrate binding-modules, share an interesting property: they are capable of reacting with free (exogenous) cobalamin as well as with enzyme-bound cobalamin.
See the MetH Movie!
This movie shows the conformational changes necessary for catalysis by cobalamin-dependent methionine synthase. This multi-domain protein consists of four modules: the green module binds homocysteine which is converted to methionine by methylation. The gold module binds methyltetrahydrofolate which provides the methyl groups. The red module binds the cobalamin (B12) cofactor which accepts methyl groups from methyltetrahydrofolate and passes them to homocysteine. You can see these modules alternating at the cobalamin site in the movie: but the real rate of methyl transfer is 29 per second! Every so often, the enzyme becomes inactivated, and then the blue module is needed to catalyze the resuscitation of the enzyme. [ Bandarian et al., (2003) Proc. Natl. Acad. Sci. (inaugural contribution of Rowena Matthews) ]
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Activation of Homocysteine
The final area in which the Matthews laboratory has made significant contributions to mechanistic enzymology is in understanding the mechanism for activation of homocysteine in both MetH and MetE. Dr. Matthews has shown that zinc is catalytically essential for both MetH and MetE. It now appears that the use of zinc to coordinate thiols that are to be alkylated may be quite common; zinc has now been found in a variety of related methyl and alkyl-transferases.
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