Abstract
Methionine synthase catalyzes a methyl transfer reaction from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate and is dependent on methylcobalamin, a derivative of vitamin B12, for activity. Due to the lability of the intermediate, cob(I)alamin, the activity of methionine synthase is additionally dependent on a redox activation system. In bacteria, two flavoproteins, NADPH-flavodoxin reductase and flavodoxin, shuttle electrons from NADPH to methionine synthase. Their mammalian counterparts are unknown, and a putative intrinsic thiol oxidase activity of the mammalian methionine synthase has been proposed to be involved. We demonstrate that the mammalian methionine synthase can be activated in an NADPH-dependent reaction and requires a minimum of two redox proteins. This model is consistent with our results from biochemical complementation studies between cblG and cblE cell lines and mutation detection analysis in cblG cell lines. These demonstrate that the cblG cell line has defects affecting methionine synthase directly, whereas the cblE cell line has defects in the redox proteins. We have also identified a P1173L mutation in the activation domain of methionine synthase in the cblG cell line WG1505.
Highlights
Methionine synthase (E.C. 2.1.1.13) is a cobalamin-dependent enzyme that catalyzes two successive transmethylation reactions [1, 2]
A thiol oxidase activity associated with crude porcine liver methionine synthase and assumed to be an intrinsic property of the enzyme has been postulated to function in reductive activation [5]
NADPH-dependent Activity of Mammalian Methionine Synthase—Thiols have been implicated as physiological reductants of the mammalian methionine synthase based on the observation that a putative thiol oxidase activity is associated with the crude enzyme [5]
Summary
Vol 272, No 31, Issue of August 1, pp. 19171–19175, 1997 Printed in U.S.A. Defects in Auxiliary Redox Proteins Lead to Functional Methionine Synthase Deficiency*. We demonstrate that the mammalian methionine synthase can be activated in an NADPH-dependent reaction and requires a minimum of two redox proteins This model is consistent with our results from biochemical complementation studies between cblG and cblE cell lines and mutation detection analysis in cblG cell lines. Fractionation of crude homogenates indicates that at least two redox proteins are required for NADPH-dependent activation of porcine methionine synthase We have exploited these observations to distinguish between the defective loci in cblG and cblE cell lines that exhibit functional methionine synthase deficiency.
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