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At least 19 recordsLinked to original sources
PubMed1990

Two distinct cDNAs for human IMP dehydrogenase.

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.

Amino Acid Sequence↗
PubMed2000

Asp338 controls hydride transfer in Escherichia coli IMP dehydrogenase.

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+). This reaction involves the formation of a covalent adduct with an active site Cys. This intermediate, E-XMP, hydrolyzes to produce XMP. The mutation of Asp338 to Ala severely impairs the activity of Escherichia coli IMPDH, decreasing the value of k(cat) by 650-fold. No (D)V(m) or (D)V/K(m) isotope effects are observed when 2-(2)H-IMP is the substrate for wild-type IMPDH. Values of (D)V(m) = 2.6 and (D)V/K(m) (IMP) = 3.4 are observed for Asp338Ala. Moreover, while a burst of NADH production is observed for wild-type IMPDH, no burst is observed for Asp338Ala. These observations indicate that the mutation has decreased the rate of hydride transfer by at least 5 x 10(3)-fold. In contrast, k(cat) for the hydrolysis of 2-chloroinosine-5'-monophosphate is decreased by only 8-fold. In addition, the rate constant for inactivation by 6-chloropurine riboside 5'-monophosphate is increased by 3-fold. These observations suggest that the mutation has little effect on the nucleophilicity of the active site Cys residue. These results are consistent with a recent crystal structure that shows a hydrogen bonding network between Asp338, the 2'-OH of IMP, and the amide group of NAD(+) [Colby, T. D., Vanderveen, K., Strickler, M. D., Markham, G. D., and Goldstein, B. M. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3531-3536].

Alkylation↗
PubMed2005

Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP dehydrogenase.

IMP dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the reduction of NAD(+). The reaction involves formation of an E-XMP covalent intermediate; hydrolysis of the E-XMP intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700-11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of K(R) ranging from 40 to 400 mM. The value of k(rescue) for the best rescue agents approaches the value of k(cat) for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that the guanidine derivatives do not restore the equilibrium between open and closed conformations. Therefore, rescue agents must accelerate the hydrolysis of the E-XMP intermediate. The rate of the rescue reaction increases with an increase in pH, consistent with the hypothesis that the reaction involves neutral guanidine. A solvent D(2)O isotope effect is observed at low concentrations of the rescue agent, consistent with rate-limiting transfer of a proton from water. The value of k(cat) (rescue)/K(R)(base) correlates with the pK(a) of the guanidine derivative (Bronsted coefficient beta approximately 1). These results suggest that proton transfer from water to guanidine is almost complete in the transition state.

Alanine↗
PubMed1990

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity.

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

Biomarkers↗
PubMed1987

A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase.

The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available.

Alcohol Dehydrogenase↗
PubMed1993

Characterization of human type I and type II IMP dehydrogenases.

Human IMP dehydrogenase, a target for anticancer and immunosuppressive chemotherapy, exists as two isoforms, types I and II. Nonfusion sequences of each isoform were overexpressed in an IMP dehydrogenase-deficient strain of Escherichia coli and purified to homogeneity. Both recombinant isoforms were tetramers, which was in agreement with the subunit structure of the native mammalian enzyme. The results of initial velocity and product inhibition studies were consistent with an Ordered Bi Bi kinetic mechanism for both isoforms. Substrate affinities were similar for types I and II with Km values of 18 and 9.3 microM, respectively, for IMP, and 46 and 32 microM, respectively, for NAD.kcat values were 1.5 and 1.3 s-1 at 37 degrees C for types I and II, respectively. Xanthosine 5'-monophosphate and NADH inhibited the two isoforms with identical inhibition patterns and inhibition constants. Mycophenolic acid, however, inhibited the type II enzyme with a 4.8-fold lower K than the type I. Selective inhibitors of the inducible type II isoform may mitigate toxicity caused by inhibition of the constitutively expressed type I isoform.

Amino Acid Sequence↗
PubMed1999

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show biochemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification of these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.

Amino Acid Sequence↗
PubMed1994

Tissue-differential expression of two distinct genes for human IMP dehydrogenase (E.C.1.1.1.205).

Human IMP dehydrogenase (E.C. 1.1.1.205) is recently regarded as a potent targeting enzyme for immunosuppressive drugs. Tissue differential expressions of human type I and type II IMP dehydrogenase were investigated in sixteen human adult organs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes) and five human fetal organs (heart, brain, lung, liver, kidney) using Northern blot analysis. In all tissues examined in this study, the sizes of mRNAs of each isoform were identical, respectively. The 2.3 kb type II mRNA was shown predominantly, and the 3.5 kb type I mRNA level was lower than type II in most human tissues examined. In contrast, type I IMPDH gene expressed higher than type II in peripheral blood leukocytes, uniquely. We also demonstrated that both type I and type II IMPDH genes are widely distributed among various species by Southern blot analysis. Interestingly, type I IMPDH gene may have multiple gene families in primates.

Adult↗
PubMed1993

IMP dehydrogenase inhibitors as immunomodulators.

IMP dehydrogenase is a key enzyme in the de novo pathway of purine biosynthesis and is responsible for catalyzing the first step in the formation of guanine ribonucleotides from inosine monophosphate. Mizoribine, an immunosuppressive agent in wide-spread clinical use in Japan, has been demonstrated to inhibit this enzyme. We have investigated the effects of mizoribine on human T cell activation. Stimulation of purified human peripheral blood T lymphocytes with phorbol ester and ionomycin leads to a five-fold increase in guanine ribonucleotide levels over 72 hours. The addition of mizoribine to these cultures at concentrations that are achieved in vivo leads to a dose-dependent inhibition of proliferation and concomitant 50% decrease in guanine ribonucleotide levels, an effect that is reversible with the addition of guanosine, which repletes the GTP pool. Similar effects are seen with direct stimulation via the CD3/T cell receptor complex. Inhibition of proliferation occurs at the G1/S interface of the cell cycle and is additive to that produced by cyclosporine. In order to determine whether inhibition of IMP dehydrogenase is a common mechanism of immunosuppression for drugs such as azathioprine and 6-mercaptopurine that interfere with purine biosynthesis, we compared the effects of these agents on the metabolism of purified T lymphocytes. The results of these studies demonstrate that mizoribine and mycophenolic acid, a highly specific inhibitor of IMP dehydrogenase, inhibit proliferation directly by the depletion of guanine ribonucleotides; 6-mercaptopurine, on the other hand, has a mixed effect on adenine and guanine ribonucleotide pools, whereas azathioprine inhibits proliferation by a mechanism completely independent of its effects on the purine metabolic pathway. We conclude from these studies that inhibitors of IMP dehydrogenase have potential as specific immunosuppressive agents.

Azathioprine↗
PubMed1991

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.

Amino Acid Sequence↗
PubMed1992

Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.

Antimetabolites, Antineoplastic↗
PubMed1980

Selective guanosine phosphate deficiency in hepatoma cells induced by inhibitors of IMP dehydrogenase.

Inhibition of IMP dehydrogenase in AS-30D hepatoma cells in suspension culture resulted in a pronounced and selective reduction of guanine nucleotide pools. Total acid-soluble guanine nucleotides decreased to 40% and the content of GTP and GDP dropped to about 20% of control within 4 h when mycophenolate or ribavirin were used as the inhibitors. Induction of GTP deficiency was associated with a 50% rise in UTP and other uracil nucleotides. Guanosine rapidly reversed both the reduction of guanine nucleotide pools and the elevation of cellular UTP contents. Enzymatic nucleotide analyses in cell and tissue extracts after treatment with ribavirin indicated that ribavirin 5'-triphosphate was an effective substrate for yeast hexokinase, yeast phosphoglycerate kinase, and nucleosidediphosphate kinase from yeast or bovine liver. These results were confirmed in detail by the use of synthetic ribavirin 5'-triphosphate and 5'-diphosphate. The latter nucleotide analog was also a substrate of pyruvate kinase from muscle. Mycophenolate-induced GTP deficiency was associated with an arrest of hepatoma cell growth in suspension culture. Ribavirin, at an equimolar concentration, was much less effective in this respect. None of the two inhibitors had a detectable effect, however, in vivo when guanine or uracil nucleotides were assayed in liver. This indicated that an inhibition of de novo guanylate synthesis in vivo can be compensated by salvage pathway synthesis.

Animals↗
PubMed2004

Detection of the mycophenolate-inhibited form of IMP dehydrogenase in vivo.

IMP dehydrogenase (IMPDH) is the rate-limiting enzyme for de novo GMP synthesis. Its activity is correlated with cell growth, and it is the target of a number of proven and experimental drug therapies including mycophenolic acid (MPA). MPA inhibits the enzyme by trapping a covalent nucleotide-enzyme intermediate. Saccharomyces cerevisiae has four IMPDH genes called IMD1-IMD4. IMD2 is transcriptionally regulated and is the only one that enables yeast to grow in the presence of MPA. We show here that de novo synthesis of the IMD2-encoded protein is strongly induced upon MPA treatment. We also monitor the in vivo formation of a covalent nucleotide-enzyme intermediate for Imd2, Imd3, and Imd4 that accumulates in the presence of MPA. Complete formation of the Imd2 intermediate requires drug concentrations manyfold higher than that required to quantitatively trap the Imd3- or Imd4-nucleotide adducts. Purification of the tagged IMD gene products reveals that the family of polypeptides coassemble to form heteromeric IMPDH complexes, suggesting that they form mixed tetramers. These data demonstrate that S. cerevisiae harbor multiple IMPDH enzymes with varying drug sensitivities and offer an assay to monitor the inhibition of IMPDH in living cells. They also suggest that mixed inhibition profiles may result from heteromeric complexes in cell types that contain multiple IMPDH gene products. The mobility shift assay could serve as a tool for the detection of drug-inactivated IMPDH in the cells of patients receiving MPA therapy.

Base Sequence↗
PubMed1989

Increased activity, amount, and altered kinetic properties of IMP dehydrogenase from mycophenolic acid-resistant neuroblastoma cells.

Mouse wild-type neuroblastoma cells (NB cells) were stepwise selected for 10,000-fold increased resistance to mycophenolic acid (NB-Myco cells), an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). IMP dehydrogenase activity was increased 25-fold, from 3.1 to 75 nmol/min.mg of protein; and a 56.7-kDa peptide was increased in abundance 200-500-fold in NB-Myco as compared to NB cells. Purification and sequence analysis confirmed that the abundant protein was IMP dehydrogenase. The stepwise selection, increased activity and protein abundance, and unstable phenotype are indirect evidence for a process of gene amplification. Kinetic findings consistent with an Ordered Bi Bi mechanism were indicative of IMP dehydrogenase having undergone mutation. The Michaelis constants were unchanged for IMP (14 and 13 microM) and increased 4-fold for NAD from 25 to 94 microM for NB and NB-Myco cells, respectively. The Ki for mycophenolic acid was increased 2400-fold from 1.4 nM to 3.4 microM for the enzyme from NB versus NB-Myco cells, and the Ki for XMP was increased 4-fold from 78 to 336 microM. Mycophenolic acid exhibited uncompetitive inhibition with IMP, consistent with the formation of a dead end E-XMP-inhibitor complex. The cellular GTP concentration was increased 2-fold in resistant cells and, upon removal of mycophenolic acid, further increased to 4.5-fold that of NB cells.

Amino Acid Sequence↗
PubMed1998

Purification and some properties of IMP dehydrogenase of Bacillus cereus.

IMP dehydrogenase was purified from a crude extract of B, cereus cells. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE and 225 kDa by gel filtration. The optimum pH of the enzyme was about 9.5. The first seven residues at N-terminus of the enzyme was determined to be Met-Trp-Glu-Ser-Lys-Phe-Val. The enzyme showed a significant specificity for inosine nucleotides among 15 purines and pyrimidines tested, but not acted on other purines and pyrimidines including inosine. Among 11 metal ions and 3 enzyme inhibitors tested, Al3+ activated the IMP dehydrogenase. The enzyme activity was strongly inhibited by Zn2+ and Fe3+.

Ammonium Sulfate↗
PubMed2000

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase.

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensitive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyzed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh gene and flanking regions was sequenced. The fragment contained an open reading frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA cloning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coli using a pET17 x b expression plasmid. The N-terminal amino acid sequence of the overproduced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-Leu-Thr-Phe-AspAsp-Val-Leu -Leu-Val- Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enzyme was active against IMP, IDP, and ITP, and showed the highest activity at pH 9.5. These properties of the recombinant enzyme were almost identical to those of IMP dehydrogenase of B. cereus.

Amino Acid Sequence↗
PubMed1983

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells.

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

[Study of the regulation of the synthesis and activity of IMP-dehydrogenase in the yeast Pichia guilliermondii].

Activity of IMP-dehydrogenase was studied in the cells of P. guilliermondii ATCC 9058 from different growth phase; the yeast was cultivated in iron-rich and iron-deficient media. The highest activity of the enzyme was found in the iron-rich cells from the logarithmic growth phase. In the iron-deficient cells synthetizing great amounts of riboflavin the IMP-dehydrogenase activity was somewhat higher in negative growth acceleration phase than that of iron-rich cells from the same stage. Guanine considerably represses synthesis of IMP-dehydrogenase in the cells of the mutant P. guilliermondii Y-2031 with the blocked GMP-synthetase. In the iron-deficient cells no changes in the character of IMP-dehydrogenase synthesis regulation were found in comparison with the iron-rich cells. 5'-GMP, 5'-GDP and 5'-GTP considerably inhibit IMP-dehydrogenase activity of the cell-free preparations of the yeast P. guilliermondii. 5'-AMP, 5'UMP and 5'-CMP slightly affect the enzyme activity. Guanine inhibits LMP-dehydrogenase activity in experimeents in vivo with the guanine- and arginine-requiring "Y" 2031/arg. Thus, synthesis of guanylic acid in the yeast P. guilliermondii is regulated by means of two mechanisms repression of IMP dehydrogenase synthesis and inhibition of the activity of this enzyme by guanyl compounds.

Ascomycota↗