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R E MacKenzie

Publications and source records attributed to R E MacKenzie.

At least 37 records · Page 2Linked to original sources
PubMed1993

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.

The recombinant human bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is unique in its absolute requirement for Mg2+ and inorganic phosphate. Both ions affect the affinity of the enzyme for NAD and have no effect on the binding of methylenetetrahydrofolate. The NAD cofactor can be replaced by NADP with a much higher KM and lower VMAX. Kinetic investigation using NADP supports the role of Mg2+ in dinucleotide binding and illustrates that the 2'-phosphate can substitute for phosphate in this process. The human NAD-dependent bifunctional enzyme has a 44% amino acid sequence identity with the dehydrogenase-cyclohydrolase domain of the yeast mitochondrial NADP-dependent trifunctional enzyme encoded by the MIS1 gene, compared to 37% identity with the corresponding domain of the cytosolic trifunctional enzyme. The sequence comparison and the kinetic properties suggest that the NAD bifunctional enzyme is the mammalian homolog of the yeast mitochondrial trifunctional enzyme, which has evolved a unique use of inorganic phosphate to change its dinucleotide specificity from NADP to NAD. Its role is proposed to be in providing formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA required for the initiation of protein synthesis in mitochondria.

Aminohydrolases↗
PubMed1993

NAD(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase: detection of the mRNA in normal murine tissues and transcriptional regulation of the gene in cell lines.

NAD(+)-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, a nuclear-encoded mitochondrial bifunctional enzyme, is detectable in extracts of immortalized and transformed cells but not in most adult tissues (Mejia, N.R. and MacKenzie, R.E. (1985) J. Biol. Chem. 260, 14616-14620). Normal tissues contain low levels of the mRNA, with the exception of thymus and especially testis which contain much higher amounts. The protein could not be detected in any of these normal tissues either by Western analysis or by enzyme activity assay. The elevated level of the mRNA in testis is not dependent on active spermatogenesis. Oncogenic transformation of NIH 3T3 fibroblasts by the Ha-ras oncogene did not significantly affect the steady state level of the dehydrogenase-cyclohydrolase mRNA. The gene in quiescent Balb/c 3T3 fibroblasts is induced by mitogens such as serum and phorbol esters and requires de novo transcription. Post-transcriptionally, the mRNA is stabilized by factors in serum such as insulin-like growth factor-1. The intracellular location of the enzyme and its regulation of expression are consistent with its proposed role in mitochondrial biogenesis.

3T3 Cells↗
PubMed1992

The NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is not expressed in Spodoptera frugiperda cells.

The insect cell line derived from Spodoptera frugiperda (Sf9) does not express the activities of the trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. The lack of synthetase activity was confirmed by the inability to incorporate radiolabeled formate into nucleotides. The cells express, instead, a Mg2+ and NAD-dependent bifunctional methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase with properties similar to the enzyme found in the mitochondria of transformed mammalian cells. In contrast, the enzyme in Sf9 cells is localized in the cytoplasm. Nutritional studies in defined medium with dialyzed serum demonstrated that the Sf9 cell does not required added purines or pyrimidines for growth. It is auxotrophic for cysteine and glycine; this latter requirement is probably due to the absence of mitochondrial serine hydroxymethyltransferase. Incorporation of labeled glycine and serine into DNA indicates that only serine is a source of one-carbon units. These results suggest that the mitochondria in Sf9 cells do not play a major role in folate-mediated metabolism.

Aminohydrolases↗
PubMed1992

Expression of human NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in Escherichia coli: purification and partial characterization.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a bifunctional enzyme synthesized as a 37-kDa precursor that is imported into the mitochondria of embryonic and transformed mammalian cells. The cDNA encoding the human bifunctional enzyme was modified to remove nucleotides corresponding to the mitochondrial targeting sequence and was subcloned into a procaryotic expression vector under the control of the T7 RNA polymerase promoter. The soluble dehydrogenase-cyclohydrolase was expressed in Escherichia coli at levels up to 150-fold higher than those found in transformed mammalian cells. Forms of the recombinant enzyme with one, three, or seven additional amino-terminal residues were purified to homogeneity and shown to have similar kinetic properties. Investigation of the absolute requirement of the enzyme for Mg2+ using fluorescence quenching indicates that this ion binds in the absence of substrates.

Amino Acid Sequence↗
PubMed1991

Transcriptional regulation of murine NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase.

The cytosolic NADP(+)-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is ubiquitously expressed in all mouse tissues and cell lines examined. Northern analyses of the RNA indicated that there is an extensive variation in the levels of mRNA in different tissues. However, the gene is refractory to induction by serum, phorbol esters or growth factors in cultured fibroblasts. The mRNA of the NADP(+)-dependent trifunctional enzyme is stabilized post-transcriptionally by insulin-like growth factor-1.

3T3 Cells↗
PubMed1991

Analysis of the promoter region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

Sequence analysis of the 5'-flanking region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) revealed several putative cis-regulatory elements. To delineate the function of these regulatory elements, various deletion mutants of the 5'-flanking region were connected to the reporter gene chloramphenicol acetyltransferase (CAT) and promoter activity was measured in transient transfection assays. Transfection experiments performed with the sequence extending from -508 to +59 produced a high-level transient expression of the CAT gene in BALB/c 3T3-SV-T2 and NIH 3T3 cells. Removal of the sequence from +16 to +59 which includes the second transcription start point at +43, a TATA-like box and 5'-untranslated sequences abolished the promoter activity. Deletion analysis of 5'-upstream sequences revealed that the region from positions -55 to +59 is sufficient to mediate a high CAT activity comparable to the level obtained with the construct -508/+59. Within this region are found a CAAT box, a TATA-like box and two putative GC boxes. A functional analysis of the promoter showed that the sequence from -55 to +59 is sufficient to respond to stimulation by serum.

Aminohydrolases↗
PubMed1991

Structural organization of the murine gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

The structural organization of the entire nuclear gene (NMDMC) encoding the mitochondrial (mt) NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme (NMDMC) was determined by analysis of clones obtained from a lambda EMBL3 murine genomic DNA library. The gene is approx. 13 kb in length and contains eight exons and seven introns. All exon/intron splice junctions follow the GT/AG rule. The amino acid presequence, which is essential for transport of the NMDMC enzyme precursor into mt, is encoded almost entirely in the first exon. Two major transcriptional start points (tsp), located 33 and 75 nucleotides upstream from the AUG start codon, were revealed by S1 nuclease mapping and RNase protection analyses. The immediate 5'-flanking region of the first exon contains one CAAT box, a TATA-like box and three sites homologous to the consensus sequence for the binding of transcription factor Sp1.

Aminohydrolases↗
PubMed1991

A pseudogene on the X chromosome for the human trifunctional enzyme MTHFD (methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase).

The human trifunctional folate-dependent protein MTHFD has been mapped to chromosome 14q24 and to the X chromosome was identified by screening an X-chromosome-specific library. Amplification by the polymerase chain reaction (PCR) and sequencing of PCR products indicate that the sequence is an intronless pseudogene.

Aminohydrolases↗
PubMed1991

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli.

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase was engineered to contain a prokaryotic ribosome binding site and was expressed under the bacteriophage T7 RNA polymerase promoter in Escherichia coli. Site-directed mutagenesis was used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase (D/C) domain as amino acid residues 1-301, and the synthetase (Syn) domain as residues 304-935. Both domains formed active enzymes thereby demonstrating their ability to fold independently. The full-length enzyme, D/C and Syn domains were expressed at levels 4-, 55- and 3-fold higher than the specific activities found in liver. Additional mutagenesis and independent expression of domains further defined the interdomain region to include amino acids 292-310. The D/C domain was purified to homogeneity by a single affinity chromatographic step, and the full-length protein in a two-step procedure. The kinetic properties of the D/C domain appear unaltered from those of the trifunctional enzyme.

Amino Acid Sequence↗
PubMed1989

An improved procedure for the purification of formiminotransferase-cyclodeaminase from pig liver. Kinetics of the transferase activity with tetrahydropteroylpolyglutamates.

Formiminotransferase-cyclodeaminase is stabilized and activated approx. 40% in the presence of low concentrations (equal or less than 0.2%) of Triton X-100, possibly because the average hydrophobicity (1.10 kcal per residue) and the frequency of large non-polar side-chains (0.34) of this protein are both somewhat higher than average. This stabilization enabled us to develop a new purification procedure for the enzyme using chromatography on Matrex Gel Orange A and heparin-Sepharose columns in the presence of Triton X-100. This procedure is easier, much more reproducible, and gives slightly higher yield than the previous method described by Drury, et al. Further investigations of the role of tetrahydropteroylpolyglutamates with formiminotransferase-cyclodeaminase reveal that the use of polyglutamylated folate substrates does not change the mechanism of the transferase reaction, but decreases the K(m) for formininoglutamate, the second substrate, more than 10-fold, bringing it closer to the expected physiological concentration.

Amino Acids↗
PubMed1989

Isolation and characterization of cDNA clones encoding the murine NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

Forty cDNA clones corresponding to the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme were isolated from a mouse lambda gt11 library. Two classes of cDNA clones were shown by Northern analysis to correspond to the two mRNA species of 1.7 and 2.0 kilobases present in transformed cells but not in normal tissues and that apparently are derived from alternate polyadenylation signals. The 1050-base pair coding region encodes a protein of 350 amino acids which contains a putative mitochondrial-targeting signal peptide of 34 amino acids following the initiator methionine. The 20 amino acids immediately following the signal peptide correspond exactly to those determined by sequence analysis of the amino terminus of the purified protein. The derived amino acid sequence of the NAD-dependent dehydrogenase-cyclohydrolase shows extensive homology with the corresponding amino-terminal sequence of the trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase enzyme from human cells (approximately 40%), yeast cytosol (approximately 36%), and yeast mitochondria (approximately 45%).

Amino Acid Sequence↗
PubMed1989

Chromosomal localization of the gene for the human trifunctional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

A trifunctional protein in man, 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of tetrahydrofolate derivatives; these derivatives supply one-carbon units for intermediary metabolism. Somatic cell hybridization and in situ hybridization were used to localize the functional gene coding for this protein--to human chromosome 14q24, near the c-fos and TGF-beta 3 loci. A second hybridizing sequence, possibly a pseudogene, was identified near the centromere of the X chromosome, at Xp11.

Aminohydrolases↗
PubMed1988

Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

A DNA clone complementary to the messenger RNA encoding the human trifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyl-tetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase has been isolated from a lambda gt10 library. In vitro transcription-translation of the 3.1-kilobase cDNA clone yields a protein of 101 kDa, which is of identical size and exhibits the same immunoreactivity as the enzyme purified from human liver. A coding region of 2805 base pairs in the cDNA encodes a protein of 935 amino acids. The initiator methionine is absent from the purified enzyme and the amino-terminal 30 amino acids derived by automated sequence analysis are identical (arginine at position 18 was not identified) with that deduced from the nucleotide sequence. The amino acid sequence of the human enzyme shows extensive homology with that of the yeast enzyme, although the amino-terminal bifunctional dehydrogenase-cyclohydrolase domain is less homologous than the carboxyl-terminal synthetase domain. A region was identified which probably serves as a link between these two major domains of the human enzyme. The synthetase domain contains two regions that are homologous to consensus sequences for an ATP-binding site.

Amino Acid Sequence↗
PubMed1988

Substrate flux through methylenetetrahydrofolate dehydrogenase: predicted effects of the concentration of methylenetetrahydrofolate on its partitioning into pathways leading to nucleotide biosynthesis or methionine regeneration.

Folic acid exists in mammalian cells with a poly-gamma-glutamate tail that may regulate the flux of folates through the various cellular pathways. The substrate polyglutamate specificity of methylenetetrahydrofolate dehydrogenase from pig liver has been examined by using a competitive method and measuring apparent tritium kinetic isotope effects on Vmax/Km for methylenetetrahydrofolate. This competitive method yields very accurate ratios of Km values for alternate substrates of an enzyme and may also be applied to reactions with no isotope effect. In combination with published data from our own and other laboratories, the kinetic parameters of methylenetetrahydrofolate dehydrogenase were used to calculate the initial velocities of pig liver methylenetetrahydrofolate dehydrogenase, thymidylate synthase, and methylenetetrahydrofolate reductase, at physiological concentrations of substrates and enzymes. These calculations suggest that the cellular concentration of methylenetetrahydrofolate may regulate the flux of this metabolite into the pathways leading to nucleotide biosynthesis and methionine regeneration. An increase in the cellular level of methylenetetrahydrofolate would permit more one-carbon units to be directed toward nucleotide biosynthesis.

Animals↗
PubMed1988

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme.

Transformed mammalian cells express a unique bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in addition to the usual NADP-dependent dehydrogenase-cyclohydrolase-synthetase. Subcellular fractionation of murine cell lines revealed that the NAD-dependent dehydrogenase activity is located predominantly in mitochondria, while the NADP-dependent trifunctional dehydrogenase appears to exist only in the cytosol of these cells. Western analysis using monospecific polyclonal antisera confirms the subcellular distribution of these two proteins.

Aminohydrolases↗
PubMed1988

Renaturation of formiminotransferase-cyclodeaminase from guanidine hydrochloride. Quaternary structure requirements for the activities and polyglutamate specificity.

Formiminotransferase-cyclodeaminase denatured in 6 M guanidine hydrochloride (Gdn.HCl) refolds and reassembles to the native octameric structure upon dilution into buffer. Both enzymic activities are recovered to greater than 90%, and the renatured enzyme "channels" the formiminotetrahydropteroylpentaglutamate intermediate. Under conditions where the two activities are recovered simultaneously, the rate-limiting step in reactivation is first order with respect to protein, with k = 1.9 X 10(-5) s-1 at 22 degrees C and delta E approximately equal to 15 kcal mol-1. In the presence of 1.5 M urea, renaturation is arrested at the level of dimers having only transferase activity. Subsequent dialysis to remove the urea leads to recovery of deaminase activity and formation of octamer. Kinetic studies with mono- and pentaglutamate derivatives of the folate substrates demonstrated that native and renatured enzyme as well as deaminase-active dimers [Findlay, W. A., & MacKenzie, R. E (1987) Biochemistry 26, 1948-1954] have much higher affinity for polyglutamate substrates, while the transferase-active dimers do not. These results indicate that the transferase activity is associated with one type of subunit-subunit interaction in the native tetramer of dimers and that the polyglutamate binding site and the deaminase activity are associated with the other interface. A dimeric transferase-active fragment generated by limited proteolysis of the native enzyme can also be renatured from 6 M Gdn.HCl, confirming that it is an independently folding domain capable of reforming one type of subunit interaction.

Ammonia-Lyases↗
PubMed1988

The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent.

The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.

Aminohydrolases↗