Methionine synthases are folate-dependent enzymes that catalyze the
transfer of a methyl group from 5-methyltetrahydropteroylpolyglutamate (CH3–
H4PteGlun), also called 5-methyltetrahydrofolate, to L-homocysteine to form L-methionine. There are two major classes of methionine synthases, the cobalamin-dependent and the cobalamin-independent methionine synthases.
The cobalamin-dependent methionine synthase is a very large, 140 kDa protein, and uses cobalamin to aid in the transfer of the methyl group from 5-
CH3–H4PteGlun to homocysteine. Only organisms that can synthesize or obtain
cobalamin, such as mammals, use cobalamin-dependent methionine synthase.
Organisms that cannot obtain or synthesize cobalamin, such as fungi, use
the cobalamin-independent methionine synthases, and some bacteria such as E.
coli use enzymes from both classes. Proteins from the cobalamin-independent class have a molecular weight of 86 kDa, and have no amino acid sequence
homology to the cobalamin-dependent enzymes. These enzymes are zinc
dependent, and kinetic analyses of the E. coli cobalamin-independent methionine
synthase (MetEp) reveal that it will only bind polyglutamated forms of 5-CH3–
H4PteGlun. Methionine synthases from fungi are not well characterized. They
may be interesting anti-fungal drug targets because of the mechanistic differences
between them and the cobalamin-dependent forms present in humans.
C. albicans resides in the normal flora of the human body. However, it is
able to cause infection in immunocompromised patients. In the past two decades,
C. albicans has become one of the most common opportunistic pathogens,
particularly in hospitals. Increasing drug resistance to present drugs, and severe
side effects results in the constant search for new drug targets to create better and
more effective therapies.
The work presented here investigates the cobalamin-independent
methionine synthase from C. albicans (CaMet6p) and from S. cerevisiae
(ScMet6p). Substrate specificity for both enzymes was explored through kinetic
analyses, and a strategy was implemented to study important active site residues
by site-directed mutagenesis. A conditional cobalamin-independent methionine
synthase (CaMET6) mutant in C. albicans was constructed, using the PCR-based
gene disruption method, to assess the viability of the resulting null mutant strain. The results from these experiments have provided new insights into enzyme
function, and support the study of CaMet6p as an anti-fungal drug target.Chemistry and BiochemistryChemistr
