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Nigel S. Scrutton - One of the best experts on this subject based on the ideXlab platform.
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Gating mechanisms for biological electron transfer: Integrating structure with biophysics reveals the nature of redox control in cytochrome P450 Reductase and copper-dependent nitrite Reductase
2020Co-Authors: Nicole G. H. Leferink, Stephen E. J. Rigby, Christopher R. Pudney, Sibylle Brenner, Derren J. Heyes, Robert R. Eady, S. Samar Hasnain, Sam Hay, Nigel S. ScruttonAbstract:Edited by Miguel Teixeira and Ricardo O. Louro Keywords: Electron transfer Gating Proton coupled electron transfer Conformationally controlled electron transfer a b s t r a c t Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 Reductase, Methionine Synthase Reductase and copper-dependent nitrite Reductase. Specifically, we review the evidence for conformational control in cytochrome P450 Reductase and Methionine Synthase Reductase and chemical control i.e. proton coupled electron transfer in nitrite Reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems
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Tryptophan 697 Modulates Hydride and Interflavin Electron Transfer in Human Methionine Synthase Reductase
Biochemistry, 2011Co-Authors: Carla E. Meints, Nigel S. Scrutton, Frida S. Gustafsson, Kirsten R. WolthersAbstract:Human Methionine Synthase Reductase (MSR), a diflavin oxidoReductase, plays a vital role in Methionine and folate metabolism by sustaining Methionine Synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supp...
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Gating mechanisms for biological electron transfer: Integrating structure with biophysics reveals the nature of redox control in cytochrome P450 Reductase and copper-dependent nitrite Reductase
FEBS letters, 2011Co-Authors: Nicole G. H. Leferink, Stephen E. J. Rigby, Christopher R. Pudney, Sibylle Brenner, Derren J. Heyes, Robert R. Eady, S. Samar Hasnain, Sam Hay, Nigel S. ScruttonAbstract:Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 Reductase, Methionine Synthase Reductase and copper-dependent nitrite Reductase. Specifically, we review the evidence for conformational control in cytochrome P450 Reductase and Methionine Synthase Reductase and chemical control i.e. proton coupled electron transfer in nitrite Reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems.
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ELDOR spectroscopy reveals that energy landscapes in human Methionine Synthase Reductase are extensively remodelled following ligand and partner protein binding.
Chembiochem : a European journal of chemical biology, 2011Co-Authors: Stephen E. J. Rigby, Kirsten R. Wolthers, Xiaodong Lou, Helen S. Toogood, Nigel S. ScruttonAbstract:modules, which bind, from the N to C terminus, respectively, homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylMethionine (SAM). The C-terminal “activation domain” (AD) also interacts with Methionine Synthase Reductase (MSR), which is an NADPH-dependent diflavin oxidoReductase containing 1 mol equivalent of FAD and FMN. Occasionally, MS becomes inactivated through oxidation of cob(I)alamin to cob(II)alamin and reactivation by electron transfer from MSR is required. Direct reduction of MS by NADPH via FAD and FMN is thermodynamically not feasible because the redox potential of cob(I/II)alamin is far more negative than that of NADP . Consequently, Nature traps the small amounts of cob(I)alamin in equilibrium with cob(II)alamin by irreversible methylation with SAM (Scheme 1). The inferred multidomain structure of MSR and the presence of a large linker between the component flavin-containing domains suggests a large degree of conformational flexibility in the protein. By analogy with other diflavin oxidoReductases related to MSR, such as nitric oxide Synthase and cytochrome P450 Reductase, it has been suggested that MSR fluctuates between “closed” and “open” conformations. In this simple model, one conformation (“open” conformation) of MSR presents the FMN domain so that it is available for interaction with the AD of MS; in another conformation (a “closed” conformation) the FMN domain is in close proximity to the NADPH/FAD-binding domain, where it facilitates interflavin electron transfer (Figure 1). Although this model has not been tested explicitly, these multiple conformations would optimise electronic coupling to the FAD domain (closed conformation) and cob(II)alamin (open conformation) and require a “swinging” motion for the FMN domain between the two states. Such motion would, therefore, define an energy landscape for the conformationally flexible MSR protein (Figure 1). In general, the exploration of energy landscapes is integral to conformational sampling mechanisms for many biological Scheme 1. The catalytic and reactivation cycles of MS. During the primary catalytic cycle MS uses enzyme-bound cob(I)alamin to abstract a methyl group from methyl tetrahydrofolate to generate tetrahydrofolate and methylcob(III)alamin. Regeneration of inactivated MS requires one electron derived from NADPH-dependent MSR and methyl transfer from SAM; SAM: S-adenosylMethionine; SAH: S-adenosylhomocysteine.
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Cobalamin uptake and reactivation occurs through specific protein interactions in the Methionine Synthase-Methionine Synthase Reductase complex.
The FEBS journal, 2009Co-Authors: Kirsten R. Wolthers, Nigel S. ScruttonAbstract:Human Methionine Synthase Reductase (MSR), a diflavin enzyme, restores the activity of human Methionine Synthase through reductive methylation of Methionine Synthase (MS)-bound cob(II)alamin. Recently, it was also reported that MSR enhances uptake of cobalamin by apo-MS, a role associated with the MSR-catalysed reduction of exogenous aquacob(III)alamin to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG (2006) Proc Natl Acad Sci USA103, 9476-9481]. Here, we report the expression and purification of human Methionine Synthase from Pichia pastoris. This has enabled us to assess the ability of human MSR and two other structurally related diflavin Reductase enzymes (cytochrome P450 Reductase and the Reductase domain of neuronal nitric oxide Synthase) to: (a) stimulate formation of holo-MS from aquacob(III)alamin and the apo-form of MS; and (b) reactivate the inert cob(II)alamin form of MS that accumulates during enzyme catalysis. Of the three diflavin Reductases studied, cytochrome P450 Reductase had the highest turnover rate (55.5 s(-1)) for aquacob(III)alamin reduction, and the Reductase domain of neuronal nitric oxide Synthase elicited the highest specificity (k(cat)/K(m) of 1.5 x 10(5) m(-1) s(-1)) and MSR had the lowest K(m) (6.6 microm) for the cofactor. Despite the ability of all three enzymes to reduce aquacob(III)alamin, only MSR (the full-length form or the isolated FMN domain) enhanced the uptake of cobalamin by apo-MS. MSR was also the only diflavin Reductase to reactivate the inert cob(II)alamin form of purified human MS (K(act) of 107 nm) isolated from Pichia pastoris. Our work shows that reactivation of cob(II)alamin MS and incorporation of cobalamin into apo-MS is enhanced through specific protein-protein interactions between the MSR FMN domain and MS.
Rima Rozen - One of the best experts on this subject based on the ideXlab platform.
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Mouse model for deficiency of Methionine Synthase Reductase exhibits short-term memory impairment and disturbances in brain choline metabolism.
The Biochemical journal, 2014Co-Authors: Nafisa M. Jadavji, Liyuan Deng, Renata H. Bahous, Olga V. Malysheva, Marilyn Grand'maison, Barry J. Bedell, Marie A. Caudill, Rima RozenAbstract:Hyperhomocysteinaemia can contribute to cognitive impairment and brain atrophy. MTRR (Methionine Synthase Reductase) activates Methionine Synthase, which catalyses homocysteine remethylation to Methionine. Severe MTRR deficiency results in homocystinuria with cognitive and motor impairments. An MTRR polymorphism may influence homocysteine levels and reproductive outcomes. The goal of the present study was to determine whether mild hyperhomocysteinaemia affects neurological function in a mouse model with Mtrr deficiency. Mtrr+/+, Mtrr+/gt and Mtrrgt/gt mice (3 months old) were assessed for short-term memory, brain volumes and hippocampal morphology. We also measured DNA methylation, apoptosis, neurogenesis, choline metabolites and expression of ChAT (choline acetyltransferase) and AChE (acetylcholinesterase) in the hippocampus. Mtrrgt/gt mice exhibited short-term memory impairment on two tasks. They had global DNA hypomethylation and decreased choline, betaine and acetylcholine levels. Expression of ChAT and AChE was increased and decreased respectively. At 3 weeks of age, they showed increased neurogenesis. In the cerebellum, mutant mice had DNA hypomethylation, decreased choline and increased expression of ChAT. Our work demonstrates that mild hyperhomocysteinaemia is associated with memory impairment. We propose a mechanism whereby a deficiency in Methionine synthesis leads to hypomethylation and compensatory disturbances in choline metabolism in the hippocampus. This disturbance affects the levels of acetylcholine, a critical neurotransmitter in learning and memory.
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Methionine Synthase Reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice.
Molecular genetics and metabolism, 2008Co-Authors: Liyuan Deng, C. Lee Elmore, Andrea K. Lawrance, Rowena G. Matthews, Rima RozenAbstract:Abstract Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine Synthase Reductase (MTRR) is required for activation of Methionine Synthase, a folate- and vitamin B 12 -dependent enzyme. A polymorphism in MTRR (p.I22M), present in the homozygous state in 25% of many populations, may increase risk for neural tube defects. To examine the impact of MTRR deficiency on early development and congenital heart defects, we used mice harboring a gene-trapped ( gt ) allele in Mtrr . Female mice ( Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt ) were mated with male Mtrr +/g mice. Reproductive outcomes and cardiac phenotype (presence of defects and myocardial thickness) were assessed at E14.5. Mtrr -deficient mothers had more resorptions and more delayed embryos per litter (resorptions per litter: 0.29±0.13; 1.21±0.41; 1.87±0.38 and delayed embryos per litter: 0.07±0.07; 0.14±0.14; 0.60±0.24 in Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt mothers respectively). Placentae of Mtrr gt/gt mothers were smaller and their embryos were smaller, with myocardial hypoplasia and a higher incidence of ventricular septal defects (VSD) per litter (0; 0.57±0.30; 1.57±0.67 in Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt groups respectively). Embryonic Mtrr gt/gt genotype was associated with reduced embryonic length, reduced embryonic and placental weight, and higher incidence of VSD, but did not affect myocardial thickness or embryonic delay. We conclude that Mtrr deficiency adversely impacts reproductive outcomes and cardiac development in mice. These findings may have implications for nutritional prevention of heart defects, particularly in women with the common MTRR polymorphism.
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Polymorphisms in Methionine Synthase Reductase and betaine-homocysteine S-methyltransferase genes: risk of placental abruption.
Molecular genetics and metabolism, 2007Co-Authors: Cande V. Ananth, Daniel Leclerc, Denise A. Elsasser, Wendy L. Kinzler, Morgan R. Peltier, Darios Getahun, Rima RozenAbstract:Abstract Objectives: Methionine Synthase Reductase (MTRR) and betaine-homocysteine S-methyltransferase (BHMT) are two enzymes that regulate homocysteine metabolism. Elevated homocysteine (hyperhomocysteinemia) is associated with adverse pregnancy outcomes and vascular disease. We assessed whether polymorphisms in MTRR (66A → G; I22M) and BHMT (742G → A; R239Q) were associated with abruption. We further evaluated whether homocysteine levels differed between cases and controls for MTRR and BHMT genotypes. Methods: Data were derived from the New Jersey Placental Abruption Study (NJ-PAS)—an ongoing, multicenter, case-control study since August 2002. Women with a clinical diagnosis of abruption were recruited as incident cases (n = 196), and controls (n = 191) were matched to cases based on maternal race/ethnicity and parity. Total plasma homocysteine concentrations were evaluated in a subset of 136 cases and 136 controls. DNA was genotyped for the MTRR and BHMT polymorphisms. Results: Frequencies of the minor allele of MTRR were 40.8% and 42.2% in cases and controls, respectively (adjusted OR 0.79, 95% CI 0.45, 1.40). The corresponding rates for BHMT were 33.9% and 31.7%, respectively (adjusted OR 1.93, 95% CI 0.99, 4.09). Distributions for the homozygous mutant form of MTRR were similar between cases and controls (OR 1.18, 95% CI 0.62, 2.24). The rate of homozygous mutant BHMT genotype was 2.8-fold (OR 2.82, 95% CI 1.84, 4.97) higher in cases than controls. Stratification of analyses based on maternal race did not reveal any patterns in association. Conclusions: In this population, there was an association between the homozygous mutant form of BHMT (742G → A) polymorphism and increased risk for placental abruption.
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Metabolic derangement of Methionine and folate metabolism in mice deficient in Methionine Synthase Reductase
Molecular genetics and metabolism, 2007Co-Authors: C. Lee Elmore, Daniel Leclerc, Rima Rozen, Erica D. Watson, Teodoro Bottiglieri, Natalia I. Krupenko, Sergey A. Krupenko, James C. Cross, Roy A. GravelAbstract:Abstract Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form Methionine. In humans, Methionine Synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of Methionine Synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying Methionine Synthase Reductase, an enzyme essential for the activity of Methionine Synthase. This model is a hypomorph, with reduced Methionine Synthase Reductase activity, thus avoiding the lethality associated with the absence of Methionine Synthase activity. Mtrr gt/gt mice have increased plasma homocyst(e)ine, decreased plasma Methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrr gt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocyst(e)inemia may be useful in understanding biological effects of elevated homocyst(e)ine.
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Effects of polymorphisms of Methionine Synthase and Methionine Synthase Reductase on total plasma homocysteine in the NHLBI Family Heart Study.
Atherosclerosis, 2003Co-Authors: Paul F. Jacques, Roy A. Gravel, Andrew G. Bostom, Jacob Selhub, Sharron Rich, R. Curtis Ellison, John H. Eckfeldt, Rima RozenAbstract:Abstract The metabolism of homocysteine requires contributions of several enzymes and vitamin cofactors. Earlier studies identified a common polymorphism of methylenetetrahydrofolate Reductase that was associated with mild hyperhomocysteinemia. Common variants of two other enzymes involved in homocysteine metabolism, Methionine Synthase and Methionine Synthase Reductase, have also been identified. Methionine Synthase catalyzes the remethylation of homocysteine to form Methionine and Methionine Synthase Reductase is required for the reductive activation of the cobalamin-dependent Methionine Synthase. The Methionine Synthase gene (MTR) mutation is an A to G substitution, 2756A→G, which converts an aspartate to a glycine codon. The Methionine Synthase Reductase gene (MTRR) mutation is an A to G substitution, 66A→G, that converts an isoleucine to a Methionine residue. To determine if these polymorphisms were associated with mild hyperhomocysteinemia, we investigated subjects from two of the NHLBI Family Heart Study field centers, Framingham and Utah. Total plasma homocysteine concentrations were determined after an overnight fast and after a 4-h Methionine load test. MTR and MTRR genotype data were available for 677 and 562 subjects, respectively. The geometric mean fasting homocysteine was unrelated to the MTR or MTRR genotype categories (AA, AG, GG). After a Methionine load, a weak positive association was observed between change in homocysteine after a Methionine load and the number of mutant MTR alleles ( P -trend=0.04), but this association was not statistically significant according to the overall F-statistic ( P =0.12). There was no significant interaction between MTR and MTRR genotype or between these genotypes and any of the vitamins with respect to homocysteine concentrations. This study provides no evidence that these common MTR and MTRR mutations are associated with alterations in plasma homocysteine.
Kirsten R. Wolthers - One of the best experts on this subject based on the ideXlab platform.
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Proximal FAD histidine residue influences interflavin electron transfer in cytochrome P450 Reductase and Methionine Synthase Reductase
Archives of biochemistry and biophysics, 2014Co-Authors: Carla E. Meints, Sarah M. Parke, Kirsten R. WolthersAbstract:Abstract Cytochrome P450 Reductase (CPR) and Methionine Synthase Reductase (MSR) transfer reducing equivalents from NADPH to FAD to FMN. In CPR, hydride transfer and interflavin electron transfer are kinetically coupled steps, but in MSR the two catalytic steps are represented by two distinct kinetic phases leading to transient formation of the FAD hydroquinone. In human CPR, His 322 forms a hydrogen-bond with the highly conserved Asp 677 , a member of the catalytic triad. The catalytic triad is present in MSR, but Ala 312 replaces the histidine residue. To examine if this structural variation accounts for differences in their kinetic behavior, reciprocal substitutions were created. Substitution of His 322 for Ala in CPR does not affect the rate of NADPH hydride transfer or the FAD redox potentials, but does impede interflavin electron transfer. For MSR, swapping Ala 312 for a histidine residue resulted in the kinetic coupling of hydride and interflavin electron transfer, and eliminated the formation of the FAD hydroquinone intermediate. For both enzymes, placement of the His residue in the active site weakens coenzyme binding affinity. The data suggest that the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH 2 to the higher potential FMN; a mechanism for this catalytic role is discussed.
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aromatic substitution of the fad shielding tryptophan reveals its differential role in regulating electron flux in Methionine Synthase Reductase and cytochrome p450 Reductase
FEBS Journal, 2013Co-Authors: Carla E. Meints, Svetlana Simtchouk, Kirsten R. WolthersAbstract:Methionine Synthase Reductase (MSR) and cytochrome P450 Reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis. MSR W697F and W697Y showed enhanced catalysis, noted by increases in kcat and k(cat)/K(m)(NADPH) for steady-state cytochrome c(3+) reduction and a 10-fold increase in the rate constant (k(obs1)) associated with hydride transfer. Elevated primary kinetic isotope effects on k(obs1) for W697F and W697Y suggest that preceding isotopically insensitive steps like displacement of W697 are less rate determining. MSR W697Y, but not MSR W697F, showed detectable formation of the disemiquinone intermediate, indicating that the polarity of the aromatic side chain influences the rate of interflavin electron transfer. By contrast, the CPR variants (W676F and W676Y) displayed modest decreases in cytochrome c(3+) reduction, a 30- and 3.5-fold decrease in the rate of FAD reduction, accumulation of a FADH2 -NADP(+) charge-transfer complex and dramatically suppressed rates of interflavin electron transfer. We conclude for MSR that hydride transfer is 'gated' by the free energy required to disrupt dispersion forces between the FAD isoalloxazine ring and W697. By contrast, the bulky indole ring of W676 accelerates catalysis in CPR by lowering the energy barrier for displacement of the oxidized nicotinamide ring coplanar with the FAD.
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Tryptophan 697 Modulates Hydride and Interflavin Electron Transfer in Human Methionine Synthase Reductase
Biochemistry, 2011Co-Authors: Carla E. Meints, Nigel S. Scrutton, Frida S. Gustafsson, Kirsten R. WolthersAbstract:Human Methionine Synthase Reductase (MSR), a diflavin oxidoReductase, plays a vital role in Methionine and folate metabolism by sustaining Methionine Synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supp...
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ELDOR spectroscopy reveals that energy landscapes in human Methionine Synthase Reductase are extensively remodelled following ligand and partner protein binding.
Chembiochem : a European journal of chemical biology, 2011Co-Authors: Stephen E. J. Rigby, Kirsten R. Wolthers, Xiaodong Lou, Helen S. Toogood, Nigel S. ScruttonAbstract:modules, which bind, from the N to C terminus, respectively, homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylMethionine (SAM). The C-terminal “activation domain” (AD) also interacts with Methionine Synthase Reductase (MSR), which is an NADPH-dependent diflavin oxidoReductase containing 1 mol equivalent of FAD and FMN. Occasionally, MS becomes inactivated through oxidation of cob(I)alamin to cob(II)alamin and reactivation by electron transfer from MSR is required. Direct reduction of MS by NADPH via FAD and FMN is thermodynamically not feasible because the redox potential of cob(I/II)alamin is far more negative than that of NADP . Consequently, Nature traps the small amounts of cob(I)alamin in equilibrium with cob(II)alamin by irreversible methylation with SAM (Scheme 1). The inferred multidomain structure of MSR and the presence of a large linker between the component flavin-containing domains suggests a large degree of conformational flexibility in the protein. By analogy with other diflavin oxidoReductases related to MSR, such as nitric oxide Synthase and cytochrome P450 Reductase, it has been suggested that MSR fluctuates between “closed” and “open” conformations. In this simple model, one conformation (“open” conformation) of MSR presents the FMN domain so that it is available for interaction with the AD of MS; in another conformation (a “closed” conformation) the FMN domain is in close proximity to the NADPH/FAD-binding domain, where it facilitates interflavin electron transfer (Figure 1). Although this model has not been tested explicitly, these multiple conformations would optimise electronic coupling to the FAD domain (closed conformation) and cob(II)alamin (open conformation) and require a “swinging” motion for the FMN domain between the two states. Such motion would, therefore, define an energy landscape for the conformationally flexible MSR protein (Figure 1). In general, the exploration of energy landscapes is integral to conformational sampling mechanisms for many biological Scheme 1. The catalytic and reactivation cycles of MS. During the primary catalytic cycle MS uses enzyme-bound cob(I)alamin to abstract a methyl group from methyl tetrahydrofolate to generate tetrahydrofolate and methylcob(III)alamin. Regeneration of inactivated MS requires one electron derived from NADPH-dependent MSR and methyl transfer from SAM; SAM: S-adenosylMethionine; SAH: S-adenosylhomocysteine.
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Cobalamin uptake and reactivation occurs through specific protein interactions in the Methionine Synthase-Methionine Synthase Reductase complex.
The FEBS journal, 2009Co-Authors: Kirsten R. Wolthers, Nigel S. ScruttonAbstract:Human Methionine Synthase Reductase (MSR), a diflavin enzyme, restores the activity of human Methionine Synthase through reductive methylation of Methionine Synthase (MS)-bound cob(II)alamin. Recently, it was also reported that MSR enhances uptake of cobalamin by apo-MS, a role associated with the MSR-catalysed reduction of exogenous aquacob(III)alamin to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG (2006) Proc Natl Acad Sci USA103, 9476-9481]. Here, we report the expression and purification of human Methionine Synthase from Pichia pastoris. This has enabled us to assess the ability of human MSR and two other structurally related diflavin Reductase enzymes (cytochrome P450 Reductase and the Reductase domain of neuronal nitric oxide Synthase) to: (a) stimulate formation of holo-MS from aquacob(III)alamin and the apo-form of MS; and (b) reactivate the inert cob(II)alamin form of MS that accumulates during enzyme catalysis. Of the three diflavin Reductases studied, cytochrome P450 Reductase had the highest turnover rate (55.5 s(-1)) for aquacob(III)alamin reduction, and the Reductase domain of neuronal nitric oxide Synthase elicited the highest specificity (k(cat)/K(m) of 1.5 x 10(5) m(-1) s(-1)) and MSR had the lowest K(m) (6.6 microm) for the cofactor. Despite the ability of all three enzymes to reduce aquacob(III)alamin, only MSR (the full-length form or the isolated FMN domain) enhanced the uptake of cobalamin by apo-MS. MSR was also the only diflavin Reductase to reactivate the inert cob(II)alamin form of purified human MS (K(act) of 107 nm) isolated from Pichia pastoris. Our work shows that reactivation of cob(II)alamin MS and incorporation of cobalamin into apo-MS is enhanced through specific protein-protein interactions between the MSR FMN domain and MS.
Ruma Banerjee - One of the best experts on this subject based on the ideXlab platform.
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Impeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and its Responsiveness to Flavin Supplementation
Biochemistry, 2008Co-Authors: Carmen Gherasim, Uzma Zaman, Ashraf S. Raza, Ruma BanerjeeAbstract:: 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.
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Polymorphic background of Methionine Synthase Reductase modulates the phenotype of a disease-causing mutation.
Human mutation, 2007Co-Authors: Carmen Gherasim, David S. Rosenblatt, Ruma BanerjeeAbstract:Methionine Synthase Reductase (MTRR) is the locus of the cblE class of inborn errors of cobalamin metabolism that is characterized by megaloblastic anemia and homocystinuria. Two highly prevalent SNPs, c.66A>G (p.Ile22Met) and c.524C>T (p.Ser175Leu), are found in the MTRR gene. On the basis of the allele frequency of these amino acids and sequence comparison with members of the same family of proteins, the p.Ile22/p.Ser175 sequence is designated as wild type. While characterizing a pathogenic Methionine Synthase Reductase (MSR) mutation, c.166G>A (p.Val56Met), we discovered an interaction between the mutation and one of the polymorphic sites. Thus, when the p.Val56Met mutant was initially expressed in the p.Ile22/p.Ser175 background, we were surprised to find that kinetically, it was virtually indistinguishable from wild-type protein. To determine if the polymorphisms interacted with the p.Val56Met mutation, it was expressed in all four possible genetic backgrounds. We found that in the p.Ile22Met background, the p.Val56Met mutation impacted the kinetics of MSR and an approximately three- to 10-fold higher concentration of the p.Ile22Met/p.Val56Met mutant was required for maximal activation of Methionine Synthase vs. the range seen with wild-type MSR variants. A comparable (three- to seven-fold) diminution in MSR activity was observed in extracts of fibroblast cells from patients carrying the p.Val56Met mutation on one MSR allele and a null mutation on the other. These results predicted that the patient allele encodes the p.Val56Met mutation and the p.Ile22Met variation, which was confirmed by sequence analysis. This study reveals how a genetic variation can modulate phenotypic expression of a disease-causing mutation.
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Human ATP:Cob(I)alamin adenosyltransferase and its interaction with Methionine Synthase Reductase.
The Journal of biological chemistry, 2004Co-Authors: Nicole A. Leal, Horatiu Olteanu, Ruma Banerjee, Thomas A. BobikAbstract:Abstract The final step in the conversion of vitamin B12 into coenzyme B12 (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min–1 mg–1, and their Km values were 6.3 and 6.9 μm for ATP and 1.2 and 1.6 μm for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human Methionine Synthase Reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was ∼4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of Methionine Synthase) indicates a dual physiological role for MSR.
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Kinetic and thermodynamic characterization of the common polymorphic variants of human Methionine Synthase Reductase.
Biochemistry, 2004Co-Authors: Horatiu Olteanu, Kirsten R. Wolthers, Nigel S. Scrutton, Andrew W. Munro, Ruma BanerjeeAbstract:Human Methionine Synthase Reductase (MSR) is a protein containing both FAD and FMN, and it reactivates Methionine Synthase that has lost activity due to oxidation of cob(I)alamin to cob(II)alamin. In this study, anaerobic redox titrations were employed to determine the midpoint reduction potentials for the flavin cofactors in two highly prevalent polymorphic variants of MSR, I22/L175 and M22/S175. The latter is a genetic determinant of plasma homocysteine levels and has been linked to premature coronary artery disease, Down's syndrome, and neural tube defects. The I22/L175 polymorphism has been described in a homocystinuric patient. Interestingly, this polymorphism is in the extended linker region between the two flavin domains, which may mediate or facilitate interaction with Methionine Synthase. In MSR I22/L175, the FMN potentials are −103 mV (oxidized/semiquinone) and −175 mV (semiquinone/hydroquinone) at pH 7.0 and 25 °C, and the corresponding FAD potentials are −252 and −285 mV, respectively. For the...
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Redundancy in the pathway for redox regulation of mammalian Methionine Synthase: reductive activation by the dual flavoprotein, novel Reductase 1.
The Journal of biological chemistry, 2003Co-Authors: Horatiu Olteanu, Ruma BanerjeeAbstract:Abstract Methionine Synthase is an essential cobalamin-dependent enzyme in mammals that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to give tetrahydrofolate and Methionine. It is oxidatively labile and requires for its sustained activity an auxiliary repair system that catalyzes a reductive methylation reaction. Genetic and biochemical studies have demonstrated that the soluble dual flavoprotein oxidoReductase, Methionine Synthase Reductase, serves as a redox partner for Methionine Synthase in an NADPH-dependent reaction. However, three reports suggest the possibility of redundancy in this redox pathway. First, a hyperhomocysteinemic patient has been reported who has an isolated functional deficiency of Methionine Synthase but appears to be distinct from the cblE and cblG classes of patients with defects in Methionine Synthase Reductase and Methionine Synthase, respectively. Second, another dual flavoprotein oxidoReductase with significant homology to Methionine Synthase Reductase, NR1, has been described recently, but its function is unknown. Third, Methionine Synthase can be activated in vitro by a two-component redox system comprised of soluble cytochrome b5 and P450 Reductase. In this study, we demonstrate a function for human NR1 in vitro. It is able to fully activate Methionine Synthase in the presence of soluble cytochrome b5 with a Vmax of 2.8 ± 0.1 μmol min–1 mg–1 protein, which is comparable with that seen with Methionine Synthase Reductase. The KactNR1 is 1.27 ± 0.16 μm, and a 20-fold higher stoichiometry of Reductase to Methionine Synthase is required for NR1 versus Methionine Synthase Reductase, suggesting that it may represent a minor pathway in the cell, assuming that the two proteins are present at similar levels.
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mutation in folate metabolism causes epigenetic instability and transgenerational effects on development
Cell, 2013Co-Authors: Nisha Padmanabhan, Dongxin Jia, Colleen Gearyjoo, Anne C Fergusonsmith, Ernest Fung, Mark Bieda, Floyd F Snyder, Roy A. GravelAbstract:The importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme Methionine Synthase Reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.
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Restricted role for Methionine Synthase Reductase defined by subcellular localization
Molecular genetics and metabolism, 2008Co-Authors: D.s. Froese, Jun Zhang, R. Dumas, W.m. Schoel, Matthias Amrein, Roy A. GravelAbstract:Methionine Synthase Reductase (MSR; gene name MTRR) is responsible for the reductive activation of Methionine Synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target the cytosol where Methionine Synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target mitochondria was also suggested by the work of Leal et al. [N.A. Leal, H. Olteanu, R. Banerjee, T.A. Bobik, Human ATP:Cob(I)alamin adenosyltransferase and its interaction with Methionine Synthase Reductase, J. Biol. Chem. 279 (2004) 47536-47542.] who showed that it can act as the reducing enzyme in combination with MMAB (ATP:Cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol, but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin.
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Metabolic derangement of Methionine and folate metabolism in mice deficient in Methionine Synthase Reductase
Molecular genetics and metabolism, 2007Co-Authors: C. Lee Elmore, Daniel Leclerc, Rima Rozen, Erica D. Watson, Teodoro Bottiglieri, Natalia I. Krupenko, Sergey A. Krupenko, James C. Cross, Roy A. GravelAbstract:Abstract Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form Methionine. In humans, Methionine Synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of Methionine Synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying Methionine Synthase Reductase, an enzyme essential for the activity of Methionine Synthase. This model is a hypomorph, with reduced Methionine Synthase Reductase activity, thus avoiding the lethality associated with the absence of Methionine Synthase activity. Mtrr gt/gt mice have increased plasma homocyst(e)ine, decreased plasma Methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrr gt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocyst(e)inemia may be useful in understanding biological effects of elevated homocyst(e)ine.
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Human Methionine Synthase Reductase is a molecular chaperone for human Methionine Synthase.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Kazuhiro Yamada, Roy A. Gravel, Tetsuo Toraya, Rowena G. MatthewsAbstract:Sustained activity of mammalian Methionine Synthase (MS) requires MS Reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a Kact value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the presence of E. coli flavodoxin and flavodoxin Reductase, which maintain the activity of E. coli MS. These results indicate that recognition of MS by their reductive partners is very strict, despite the high homology between MS from different species. The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS). In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced. The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37°C. MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme. Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin Reductase, rather than acting solely in the reductive activation of MS.
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Effects of polymorphisms of Methionine Synthase and Methionine Synthase Reductase on total plasma homocysteine in the NHLBI Family Heart Study.
Atherosclerosis, 2003Co-Authors: Paul F. Jacques, Roy A. Gravel, Andrew G. Bostom, Jacob Selhub, Sharron Rich, R. Curtis Ellison, John H. Eckfeldt, Rima RozenAbstract:Abstract The metabolism of homocysteine requires contributions of several enzymes and vitamin cofactors. Earlier studies identified a common polymorphism of methylenetetrahydrofolate Reductase that was associated with mild hyperhomocysteinemia. Common variants of two other enzymes involved in homocysteine metabolism, Methionine Synthase and Methionine Synthase Reductase, have also been identified. Methionine Synthase catalyzes the remethylation of homocysteine to form Methionine and Methionine Synthase Reductase is required for the reductive activation of the cobalamin-dependent Methionine Synthase. The Methionine Synthase gene (MTR) mutation is an A to G substitution, 2756A→G, which converts an aspartate to a glycine codon. The Methionine Synthase Reductase gene (MTRR) mutation is an A to G substitution, 66A→G, that converts an isoleucine to a Methionine residue. To determine if these polymorphisms were associated with mild hyperhomocysteinemia, we investigated subjects from two of the NHLBI Family Heart Study field centers, Framingham and Utah. Total plasma homocysteine concentrations were determined after an overnight fast and after a 4-h Methionine load test. MTR and MTRR genotype data were available for 677 and 562 subjects, respectively. The geometric mean fasting homocysteine was unrelated to the MTR or MTRR genotype categories (AA, AG, GG). After a Methionine load, a weak positive association was observed between change in homocysteine after a Methionine load and the number of mutant MTR alleles ( P -trend=0.04), but this association was not statistically significant according to the overall F-statistic ( P =0.12). There was no significant interaction between MTR and MTRR genotype or between these genotypes and any of the vitamins with respect to homocysteine concentrations. This study provides no evidence that these common MTR and MTRR mutations are associated with alterations in plasma homocysteine.