Dataset Information

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

ABSTRACT: Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinii DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinii DHFR and, in fact, is a more potent inhibitor of human DHFR.

SUBMITTER: Edman JC

PROVIDER: S-EPMC298340 | biostudies-other | 1989 Nov

REPOSITORIES: biostudies-other

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Publications

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

Edman J C JC Edman U U Cao M M Lundgren B B Kovacs J A JA Santi D V DV

Proceedings of the National Academy of Sciences of the United States of America 19891101 22

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinii DHFR in heterologous hosts provid ...[more]

PMID: 2682653

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Project description:To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N(6)-methyl-N(6)-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N(6)-methyl-N(6)-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N(6)-methyl-N(6)-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 24) are reported as ternary complexes with NADPH. This series of bicyclic pyrido[2,3-d]pyrimidines were designed in which there was a transposition of the 5-methyl group of PTX to the N9 position of the pyrido[2,3-d]pyrimidine. It was hypothesized that the N9-methyl group would preferentially interact with Ile123 of pcDHFR (and Ile123 of pjDHFR), but not with the shorter Val115 in hDHFR. Structure-activity data for this series of antifolates revealed that a trifluoro derivative (26) was the most selective against pjDHFR compared with mammalian DHFR (h/pj = 35.7). Structural data for the hDHFR-26 complex revealed that 26 binds in a different conformation from that observed in the pcDHFR-26 complex. In the hDHFR-26 complex the trifluorophenyl ring of 26 occupies a position near the cofactor-binding site, with close intermolecular contacts with Asp21, Ser59 and Ile60, whereas this ring in the pcDHFR-26 complex is positioned away from the cofactor site and near Ile65, with weaker contacts with Ile65, Phe69 and Ile123. Comparison of the intermolecular contacts between the N9-methyl group with Val115/Ile123 validates the hypothesis that the N9-methyl substituent preferentially interacts with Ile123 compared with Val115 of hDHFR, as the weaker contact with Val115 in the hDHFR structure is consistent with its weaker binding affinity compared with pcDHFR. The results for the structures of hDHFR-22 and pcDHFR-24 show that their inhibitor-binding orientation is similar to that observed in pcDHFR-26 and the pcDHFR variant (F69N) reported previously. The naphthyl moiety of 24 makes several intermolecular contacts with the active-site residues in pcDHFR that help to stabilize the binding, resulting in a more potent inhibitor.

Project description:BackgroundThe relationship between the parasitic fungus Pneumocystis carinii and its host, the laboratory rat, presumably involves features that allow the fungus to circumvent attacks by the immune system. It is hypothesized that the major surface glycoprotein (MSG) gene family endows Pneumocystis with the capacity to vary its surface. This gene family is comprised of approximately 80 genes, which each are approximately 3 kb long. Expression of the MSG gene family is regulated by a cis-dependent mechanism that involves a unique telomeric site in the genome called the expression site. Only the MSG gene adjacent to the expression site is represented by messenger RNA. Several P. carinii MSG genes have been sequenced, which showed that genes in the family can encode distinct isoforms of MSG. The vast majority of family members have not been characterized at the sequence level.ResultsThe first 300 basepairs of MSG genes were subjected to analysis herein. Analysis of 581 MSG sequence reads from P. carinii genomic DNA yielded 281 different sequences. However, many of the sequence reads differed from others at only one site, a degree of variation consistent with that expected to be caused by error. Accounting for error reduced the number of truly distinct sequences observed to 158, roughly twice the number expected if the gene family contains 80 members. The size of the gene family was verified by PCR. The excess of distinct sequences appeared to be due to allelic variation. Discounting alleles, there were 73 different MSG genes observed. The 73 genes differed by 19% on average. Variable regions were rich in nucleotide differences that changed the encoded protein. The genes shared three regions in which at least 16 consecutive basepairs were invariant. There were numerous cases where two different genes were identical within a region that was variable among family members as a whole, suggesting recombination among family members.ConclusionA set of sequences that represents most if not all of the members of the P. carinii MSG gene family was obtained. The protein-changing nature of the variation among these sequences suggests that the family has been shaped by selection for protein variation, which is consistent with the hypothesis that the MSG gene family functions to enhance phenotypic variation among the members of a population of P. carinii.

Dataset Information

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

Publications

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

Similar Datasets

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