Impeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and Its Responsiveness to Flavin Supplementation

Abstract

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.Methionine metabolism in mammals furnishes cells with three important metabolites, Sadenosylmethionine, needed for methylation reactions, glutathione, an important antioxidant, and taurine, an osmolyte. Homocysteine is a junction intermediate in this metabolic network and, at elevated levels, constitutes a risk factor for cardiovascular diseases, neural tube defects and Alzheimer's disease (1-3). Homocysteine concentrations are kept low by the concerted actions of at least three enzymes: betaine homocysteine methyltransferase, methionine synthase, and cystathionine beta-synthase. Impairments in the latter two enzymes and in the auxiliary protein that supports the activity of methionine synthase, i.e. methionine synthase reductase (MSR) 1 , result in inborn errors of metabolism associated with severely elevated plasma homocysteine levels (4,5).

Kinetics of Reversible Reductive Carbonylation of Heme in Human Cystathionine β-Synthase

“…The absorption spectrum of MSR exhibited maxima at 380 nm and 454 nm and a shoulder at 480 nm. The ratio of A 280 /A 454 , was ~ 5.5, similar to that reported previously (36). Reduction of MSR by NADPH under aerobic conditions led to a decrease in absorbance at 454 nm and the appearance of a small shoulder in the vicinity of 585 nm and 640 nm, consistent with the formation of an air stable semiquinone.…”

“…Recombinant full-length human CBS, the truncated ΔC143 variant and MSR were purified as described previously (12, 33, 36). CBS activity was measured using either the ninhydrin method (37) or the radiolabel assay (12) as described.…”

Glutathione-dependent One-electron Transfer Reactions Catalyzed by a B12 Trafficking Protein

“…The k cat values for the ceMSR-catalyzed reduction of cytochrome c and free OH 2 Cbl at 37°C were estimated to be 1.15 Ϯ 0.07 s Ϫ1 and 40 Ϯ 4 min Ϫ1 , respectively. These activities are 3-5-fold lower than the values reported for hMSR (18,19).…”

“…To assess whether the orthologous difla- vin oxidoreductase can serve an equivalent function for ceCblC, the gene encoding ceMSR was cloned, and the protein was purified. ceMSR was judged to be active using the cytochrome c or OH 2 Cbl reduction assay in the presence of 0.2 mM NADPH as described previously (18,19). ceMSR was more prone to losing activity following dilution than hMSR, presumably due to loss of the flavin cofactor.…”

“…Reduction of cytochrome c (60 M) was performed with 50 -150 nM ceMSR in 50 mM Tris-HCl (pH 7.5) and 0.2 mM NADPH at 37°C. Reactions were monitored by the increase in absorbance at 550 nm (⌬⑀ ϭ 21 mM Ϫ1 cm Ϫ1 ), as described previously (19).…”