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 hyperhomocysteinemia may be useful in understanding biological effects of elevated homocyst(e)ine.

NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP ؉ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP ؉ . Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k cat /K m ) with NADH. Inhibition by NADP ؉ was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wildtype reductase. Both proteins were 2-3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/ W677A protein increased the sensitivity to dimethylnitrosamine by ϳ2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is the electron-donating partner for the cytochromes P-450, squalene monooxygenase, and heme oxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase is essential to cholesterol and steroid synthesis, and its importance is underscored by the developmental abnormalities and embryonic lethality observed in P-450 reductase-null mice (1). P-450 reductase contains 1 mol of FAD/ mol of enzyme and 1 mol of FMN/mol of enzyme. Reduced NADPH transfers two electrons as a hydride ion to the FAD of the enzyme, and FMN then accepts single electrons from the FAD and acts as the exit point to the protein acceptor such as the cytochromes P-450. P-450 reductase is a multi-domain protein, with the FAD and NADP(H) domains homologous with ferredoxin-NADP ϩ reductase and the FMN domain homologous with flavodoxin, suggesting that P-450 reductase was formed from an ancestral gene fusion event (2, 3). A fourth domain acts as a hinge to orient the two flavin-containing domains for electron transfer, and an NH 2 -terminal membrane-binding domain anchors the protein to the cytosolic side of the endoplasmic reticulum. The NADP(H) domain adopts the typical dinucleotide fold structure consisting of alternating ␣-helices and ␤-strands, with the nicotinamide cofactor binding in a cleft between the FAD and NADP(H) domains (4).Although expression of mammalian P-450/reductase systems in bacteria has been shown to be a valuable research tool and has promise as a tool for biodegradation (...

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. Mtrrdeficient 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.

There are now four genetic mouse models that induce hyperhomocyst(e)inemia by decreasing the activity of an enzyme involved in homocysteine metabolism: cystathionine β-synthase, methylenetetrahydrofolate reductase, methionine synthase and methionine synthase reductase. While each enzyme deficiency leads to murine hyperhomocyst(e)inemia, the accompanying metabolic profiles are significantly and often unexpectedly, different. Deficiencies in cystathionine β-synthase lead to elevated plasma methionine, while deficiencies of the remaining three enzymes lead to hypomethioninemia. The liver [S-adenosylmethionine]:[S-adenosylhomocysteine] ratio is decreased in mice lacking methylenetetrahydrofolate reductase or cystathionine β-synthase, but unexpectedly increased in mice with deficiencies in methionine synthase or methionine synthase reductase. Folate pool imbalances are seen in complete methylenetetrahydrofolate reductase deficiency, where methyltetrahydrofolate is a minor component, and in methionine synthase reductase deficiency, where methyltetrahydrofolate is increased relative to wild-type mice. These differences illustrate the potential diversity among human patients with hyperhomocyst(e)inemia, and strengthen the argument that the pathologies associated with the dissimilar forms of the condition will require different treatments.

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Mouse models that perturb homocysteine metabolism, including genetic mouse models that result in deficiencies of methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and cystathionine β-synthase, and a pharmaceutically induced mouse model with a transient deficiency in betaine-homocysteine methyl transferase, have now been characterized and can be compared. Although each of these enzyme deficiencies is associated with moderate to severe hyperhomocyst(e)inemia, the broader metabolic profiles are profoundly different. In particular the various models differ in the degree to which tissue ratios of S-adenosylmethionine to S-adenosylhomocysteine are lowered in the face of elevated plasma homocyst(e)ine, and in the distribution of the tissue folate pools. These different metabolic profiles illustrate the potential complexities of hyperhomocyst(e)inemia in humans, and suggest that comparison of the disease phenotypes of the various mouse models may be extremely useful in dissecting the underlying risk factors associated with human hyperhomocyst(e)inemia.

Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway. The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and ␣-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C. Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C␦ similarly increased activity by more than 2-fold. Addition of protein phosphatase 1␥, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo. To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet. Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C␦ with ATP, suggesting that dietary cholesterol reduced the expression of this protein. Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression. Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.

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. Methionine synthase reductase (MSR) is absolutely required for methionine synthase activity in vivo. MSR is a dual flavoprotein, containing one mol of FAD and one mol of FMN per mol of enzyme. While free FMN is highly fluorescent, enzyme‐bound FMN fluorescence is quenched. Flavin mononucleotide (FMN) binding in MSR is weak compared to other dual flavin‐containing enzymes (Kd = ~85 μM; 0 mM Pi), especially in the presence of phosphate (Kd = ~450 μM; 50 mM Pi) at constant ionic strength. Release of FMN from MSR is associated with loss of enzyme activity, as determined by cytochrome c reductase assay, while FMN‐reconstituted enzyme regains all activity. We found that the presence of 50 mM phosphate increased the overall loss of reductase activity upon dilution by ~50% and decreased the rate of rebinding of FMN ~5‐fold compared to phosphate free buffer. These findings point to a mechanism by which MSR may be inactivated under conditions that limit the availability of FMN in vivo in a phosphate sensitive manner.