Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia

Low folate intake as well as alterations in folate metabolism as a result of polymorphisms in the enzyme methylenetetrahydrofolate reductase (MTHFR) have been associated with an increased incidence of neural tube defects, vascular disease, and some cancers. Polymorphic variants of MTHFR lead to enhanced thymidine pools and better quality DNA synthesis that could afford some protection from the development of leukemias, particularly those with translocations. We now report associations of MTHFR polymorphisms in three subgroups of pediatric leukemias: infant lymphoblastic or myeloblastic leukemias with MLL rearrangements and childhood lymphoblastic leukemias with either TEL-AML1 fusions or hyperdiploid karyotypes. Pediatric leukemia patients (n = 253 total) and healthy newborn controls (n = 200) were genotyped for MTHFR polymorphisms at nucleotides 677 (C→T) and 1,298 (A→C). A significant association for carriers of C677T was demonstrated for leukemias with MLL translocations (MLL+, n = 37) when compared with controls [adjusted odd ratios (OR) = 0.36 with a 95% confidence interval (CI) of 0.15–0.85; P = 0.017]. This protective effect was not evident for A1298C alleles (OR = 1.14). In contrast, associations for A1298C homozygotes (CC; OR = 0.26 with a 95% CI of 0.07–0.81) and C677T homozygotes (TT; OR = 0.49 with a 95% CI of 0.20–1.17) were observed for hyperdiploid leukemias (n = 138). No significant associations were evident for either polymorphism with TEL-AML1+ leukemias (n = 78). These differences in allelic associations may point to discrete attributes of the two alleles in their ability to alter folate and one-carbon metabolite pools and impact after DNA synthesis and methylation pathways, but should be viewed cautiously pending larger follow-up studies. The data provide evidence that molecularly defined subgroups of pediatric leukemias have different etiologies and also suggest a role of folate in the development of childhood leukemia.

Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults

Reduction of 5,10-methylenetetrahydrofolate (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofolate (methylTHF), the primary methyl donor for methionine synthesis, is catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR). A common 677 C → T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of methylTHF and increases the pool of methyleneTHF. Recently, another polymorphism in MTHFR (1298 A → C) has been identified that also results in diminished enzyme activity. We tested whether carriers of these variant alleles are protected from adult acute leukemia. We analyzed DNA from a case–control study in the United Kingdom of 308 adult acute leukemia patients and 491 age- and sex-matched controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. The MTHFR 677TT genotype was lower among 71 acute lymphocytic leukemia (ALL) cases compared with 114 controls, conferring a 4.3-fold decrease in risk of ALL [odds ratio (OR = 0.23; 95% CI = 0.06–0.81]. We observed a 3-fold reduction in risk of ALL in individuals with the MTHFR 1298AC polymorphism (OR = 0.33; 95% CI = 0.15–0.73) and a 14-fold decreased risk of ALL in those with the MTHFR 1298CC variant allele (OR = 0.07; 95% CI = 0.00–1.77). In acute myeloid leukemia, no significant difference in MTHFR 677 and 1298 genotype frequencies was observed between 237 cases and 377 controls. Individuals with the MTHFR 677TT, 1298AC, and 1298CC genotypes have a decreased risk of adult ALL, but not acute myeloid leukemia, which suggests that folate inadequacy may play a key role in the development of ALL.

A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells

A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC 1.7.99.5), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is in vivo impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates.

Crystal structure of 2,5-diketo-d-gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

The three-dimensional structure of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo–keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of l-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-l-gulonate from d-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady–state kinetic parameters for the reassembled mutant (Phe-31 → Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein–protein interactions, which we illustrate with two examples: ras–GTPase and raf–ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.

Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y⋅) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to ≈90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 μmol⋅min−1⋅mg−1 and of (His)6-Y2 [(His)6-Y2-K387N] from yeast is 0.037 μmol⋅min−1⋅mg−1 (0.125 μmol⋅min−1⋅mg−1). Y2, Y3, and Y4 isolated from E. coli have no measurable activity. Efforts to generate Y⋅ in Y2 or Y4 using Fe2+, O2, and reductant have been unsuccessful. However, preliminary studies show that incubation of Y4 and Fe2+ with inactive E. coli Y2 followed by addition of O2 generates Y2 with a specific activity of 0.069 μmol⋅min−1⋅mg−1 and a Y⋅. A similar experiment with (His)6-Y2-K387N, Y4, O2, and Fe2+ results in an increase in its specific activity to 0.30 μmol⋅min−1⋅mg−1. Studies with antibodies to Y4 and Y2 reveal that they can form a complex in vivo. Y4 appears to play an important role in diiron-Y⋅ assembly of Y2.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by 1H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo

A gene homologous to methionine sulfoxide reductase (msrA) was identified as the predicted ORF (cosmid 9379) in chromosome V of Saccharomyces cerevisiae encoding a protein of 184 amino acids. The corresponding protein has been expressed in Escherichia coli and purified to homogeneity. The recombinant yeast MsrA possessed the same substrate specificity as the other known MsrA enzymes from mammalian and bacterial cells. Interruption of the yeast gene resulted in a null mutant, ΔmsrA::URA3 strain, which totally lost its cellular MsrA activity and was shown to be more sensitive to oxidative stress in comparison to its wild-type parent strain. Furthermore, high levels of free and protein-bound methionine sulfoxide were detected in extracts of msrA mutant cells relative to their wild-type parent cells, under various oxidative stresses. These findings show that MsrA is responsible for the reduction of methionine sulfoxide in vivo as well as in vitro in eukaryotic cells. Also, the results support the proposition that MsrA possess an antioxidant function. The ability of MsrA to repair oxidative damage in vivo may be of singular importance if methionine residues serve as antioxidants.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase

Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5′-phosphosulfate (APS) (5′-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. Further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5′-adenosinemonophosphate (5′-AMP). 2′-AMP, or 3′-phosphoadenosine-5′-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.

Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase

Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.

The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T½ cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T½ derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.