Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana

Collart, F.; Osipiuk, J.; Trent, Jonathan D.; Olsen, Gary J.; Huberman, Eliezer

doi:10.1016/0378-1119(96)00045-5

Cited by 15 publications

(11 citation statements)

References 14 publications

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“…In a microdilution assay, we found strain-specific differences in the sensitivity to MPA. (14), Homo sapiens (Human2; P12268) (39), L. donovani (Leido; P21620) (54), Arabidopsis thaliana (Arath; P47996) (12), E. coli (Ecoli; P06981) (49), and P. furiosus (Pyrfu; P42851) (11). The predicted amino acid sequences were aligned by the PILEUP program of the Wisconsin Package, version 8, using default parameters (19).…”

Section: Resultsmentioning

confidence: 99%

“…This conversion of IMP to XMP is the rate-limiting step in the biosynthetic pathway of guanine nucleotides, at least in mammals (26). While genes encoding IMP dehydrogenases have been isolated from archaea (11), protozoa (5,54,55), bacteria (2,3,16,31,36,47,49), plants (12), invertebrates (46), and mammals (10,13,39,50), the only fungal sequences known are those encoding the putative IMP dehydrogenase genes sequenced in the Saccharomyces cerevisiae genome project (14,27,28). The polymorphic fungus Candida albicans is an opportunistic pathogen in immunocompromised individuals, causing oral and vaginal candidiasis as well as disseminated infections in neutropenic patients.…”

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confidence: 99%

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Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid

1997

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An IMP dehydrogenase gene was isolated from Candida albicans on a ϳ2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomyces cerevisiae and various other organisms. Like the S. cerevisiae IMH3 sequence characterized in the genome sequencing project, the open reading frame of the C. albicans IMH3 gene is interrupted by a small intron (248 bp) with typical exon-intron boundaries and a consensus S. cerevisiae branchpoint sequence. IMP dehydrogenase mRNAs are detected in both the yeast and hyphal forms of C. albicans as judged by Northern hybridization. Growth of wild-type (sensitive) C. albicans cells is inhibited at 1 g of mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, per ml, whereas transformants hosting a plasmid with the IMH3 gene are resistant to MPA levels of up to at least 40 g/ml. The resistance of cells to MPA is gene dosage dependent and suggests that IMH3 can be used as a dominant selection marker in C. albicans.IMP dehydrogenase (EC 1.1.1.205) is the key enzyme in the de novo biosynthesis of GMP, catalyzing the NAD-dependent oxidation of IMP to XMP. This conversion of IMP to XMP is the rate-limiting step in the biosynthetic pathway of guanine nucleotides, at least in mammals (26). While genes encoding IMP dehydrogenases have been isolated from archaea (11), protozoa (5, 54, 55), bacteria (2,3,16,31,36,47,49), plants (12), invertebrates (46), and mammals (10, 13, 39, 50), the only fungal sequences known are those encoding the putative IMP dehydrogenase genes sequenced in the Saccharomyces cerevisiae genome project (14,27,28). The polymorphic fungus Candida albicans is an opportunistic pathogen in immunocompromised individuals, causing oral and vaginal candidiasis as well as disseminated infections in neutropenic patients. Genetic manipulations of this diploid microorganism have been hampered by the apparent absence of a haploid phase and the dependence of marker systems on the spontaneous occurrence of auxotrophic mutants. Thus, the identification of the IMH3 gene in C. albicans suggested to us that mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, might be used to develop a dominant selectable marker system in Candida. In several other organisms, it has been shown that amplification of an IMP dehydrogenase gene can confer MPA resistance in a gene-dosage-dependent manner by overexpression of the enzyme (9,20,23,25,54). We report the cloning and sequencing of a DNA fragment of C. albicans with a coding region for a protein with high amino acid similarity and identity to known IMP dehydrogenases. Transformation of C.albicans by using a multicopy plasmid vector yielded transformants resistant to the specific IMP dehydrogenase inhibitor MPA. Culturing of transformant cells in the presence of MPA stabilized replicative plasmids, suggesting the feasibility of using IMH3 as a dominant selection marker for genetic studies in C. alb...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid

1997

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“…Three of the identified proteins that belong to the nitrilase superfamily (nitrilases 1 and 3, and b-ureidopropionase) contain catalytic Cys (Pace and Brenner, 2001), which would be susceptible to inactivation by thiolation. Similarly, cinnamoyl CoA reductase (Goffner et al, 1994;Lacombe et al, 1997) and inosine monophosphate dehydrogenase (Collart et al, 1996) have activesite Cys that would be preferentially thiolated.…”

Section: Discussionmentioning

confidence: 99%

Stress-Induced Protein S-Glutathionylation in Arabidopsis

et al. 2005

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S-Glutathionylation (thiolation) is a ubiquitous redox-sensitive and reversible modification of protein cysteinyl residues that can directly regulate their activity. While well established in animals, little is known about the formation and function of these mixed disulfides in plants. After labeling the intracellular glutathione pool with [ 35 S]cysteine, suspension cultures of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and matrixassisted laser desorption/ionization time of flight mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation as well as proteins complexed with thiolated polypeptides. The mechanism of thiolation of five proteins, dehydroascorbate reductase (AtDHAR1), zeta-class glutathione transferase (AtGSTZ1), nitrilase (AtNit1), alcohol dehydrogenase (AtADH1), and methionine synthase (AtMetS), was studied using the respective purified recombinant proteins. AtDHAR1, AtGSTZ1, and to a lesser degree AtNit1 underwent spontaneous thiolation with GSSG-biotin through modification of active-site cysteines. The thiolation of AtADH1 and AtMetS required the presence of unidentified Arabidopsis proteins, with this activity being inhibited by S-modifying agents. The potential role of thiolation in regulating metabolism in Arabidopsis is discussed and compared with other known redox regulatory systems operating in plants.The tripeptide glutathione (GSH; g-Glu-Cys-Gly) serves important functions in plants as a reductant, transiently accumulating under stress conditions as its oxidized disulfide (GSSG). As well as forming disulfides with itself, GSH can also form mixed disulfides with proteinaceous Cys. This S-glutathionylation of proteins is commonly termed thiolation and has recently become established as a widespread reversible posttranslational modification of proteins that occurs in animal and fungal cells exposed to oxidative stress (Klatt and Lamas, 2000). Although originally considered to be a protective mechanism to prevent the irreversible oxidation of reactive protein-bound Cys to their sulfinic and sulfonic acid derivatives, it is now recognized that thiolation can act as a redox-driven regulator of signal transduction cascades and metabolic pathways (Fratelli et al., 2004). As such, thiolation has clear parallels with redox regulation of proteins mediated through intramolecular or intermolecular disulfide formation with other proteinaceous Cys (Lee et al., 2004). In plants, proteomic approaches have focused upon the redox regulation of proteins forming protein disulfides...

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“…The Arabidopsis gene encoding this enzyme was recently cloned and found to be 69% similar to human Type II IMPDH after allowing for conservative substitutions Collart et al (1996). This enzyme is of particular interest due to its role in the production of ureides in legumes (see Purine nucleotide catabolism section).…”

Section: De Novo Purine Nucleotide Metabolismmentioning

confidence: 99%