Page 21851, Table III: The kcat value for the H185A mutant was incorrectly printed. It should read 1.7 ± 0.1. The correct table is shown here.Table IIISteady-state kinetic analysis for mutant pfDHODHspfDHODHkcatKm (CoQD)Km(L-DHO)s−1μmμmWT7.8 ± 0.216 ± 191 ± 5R265A4.6 ± 0.132 ± 5105 ± 6H185A1.7 ± 0.166 ± 8123 ± 20 Open table in a new tab
S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that is processed from a single polypeptide into two subunits creating the cofactor. In the human enzyme, both the proenzyme processing reaction and enzyme activity are stimulated by the polyamine putrescine. The processing reaction of Trypanosoma cruzi AdoMetDC was studied in an in vitro translation system. The enzyme was fully processed in the absence of putrescine, and the rate of this reaction was not stimulated by addition of the polyamine. Residues in the putrescine binding site of the human enzyme were evaluated for their role in processing of the T. cruzi enzyme. The E15A, I80K/S178E, D174A, and E256A mutant T. cruzi enzymes were fully processed. In contrast, mutation of R13 to Leu (the equivalent residue in the human enzyme) abolished processing of the T. cruzi enzyme, demonstrating that Arg at position 13 is a major determinant for proenzyme processing in the parasite enzyme. This amino acid change is a key structural difference that is likely to be a factor in the finding that putrescine has no role in processing of the T. cruzi enzyme. In contrast, the activity of T. cruzi AdoMetDC is stimulated by putrescine. Equilibrium sedimentation experiments demonstrated that putrescine does not alter the oligomeric state of the enzyme. The putrescine binding constant for binding to the T. cruzi enzyme (Kd = 150 μM) was measured by a fluorescence assay and by ultrafiltration with a radiolabeled ligand. The mutant T. cruzi enzyme D174V no longer binds putrescine, and is not activated by the diamine. In contrast, mutation of E15, S178, E256, and I80 had no effect on putrescine binding. The kcat/Km values for E15A and E256A mutants were stimulated by putrescine to a smaller extent than the wild-type enzyme (2- and 4-fold vs 11-fold, respectively). These data suggest that the putrescine binding site on the T. cruzi enzyme contains only limited elements (D174) in common with the human enzyme and that the diamine plays different roles in the function of the mammalian and parasite enzymes.
Malaria is one of the most serious global infectious diseases. The pyrimidine biosynthetic enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is an important target for antimalarial chemotherapy. We describe a detailed analysis of protein-ligand interactions between DHODH and a triazolopyrimidine-based inhibitor series to explore the effects of fluorine on affinity and species selectivity. We show that increasing fluorination dramatically increases binding to mammalian DHODHs, leading to a loss of species selectivity. Triazolopyrimidines bind Plasmodium and mammalian DHODHs in overlapping but distinct binding sites. Key hydrogen-bond and stacking interactions underlying strong binding to PfDHODH are absent in the mammalian enzymes. Increasing fluorine substitution leads to an increase in the entropic contribution to binding, suggesting that strong binding to mammalian DHODH is a consequence of an enhanced hydrophobic effect upon binding to an apolar pocket. We conclude that hydrophobic interactions between fluorine and hydrocarbons provide significant binding energy to protein-ligand interactions. Our studies define the requirements for species-selective binding to PfDHODH and show that the triazolopyrimidine scaffold can alternatively be tuned to inhibit human DHODH, an important target for autoimmune diseases.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Polyamines are small organic cations found in all cells, and the biosynthetic pathway is well described in eukaryotes and Escherichia coli. The characterized pathway uses decarboxylated S-adenosylmethionine as the aminopropyl group donor to form spermidine from putrescine by the key enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We report here the in vivo characterization of an alternative polyamine biosynthetic pathway from Vibrio cholerae, the causative agent of human cholera. The pathway uses aspartate beta-semialdehyde as the aminopropyl group donor and consists of a fused protein containing l-2,4-diaminobutyrate aminotransferase and l-2,4-diaminobutyrate decarboxylase, a carboxynorspermidine dehydrogenase (CANSDH), and a carboxynorspermidine decarboxylase (CANSDC). We show that in V. cholerae, this pathway is required for synthesis of both sym-norspermidine and spermidine. Heterologous expression of the V. cholerae pathway in E. coli results in accumulation of the nonnative polyamines diaminopropane and sym-norspermidine. Genetic deletion of the V. cholerae CANSDC led to accumulation of carboxynorspermidine, whereas deletion of either CANSDC or the putative CANSDH led to loss of sym-norspermidine and spermidine. These results allowed unambiguous identification of the gene encoding CANSDH. Furthermore, deletion of either CANSDH or CANSDC led to a 50-60% reduction in growth rate of planktonic cells and severely reduced biofilm formation, which could be rescued by exogenously supplied sym-norspermidine but not spermidine. The pathway was not required for infectivity in a mouse model of V. cholerae infection. Notably, the alternative polyamine biosynthetic pathway is widespread in bacteria and is likely to play a previously unrecognized role in the biology of these organisms.
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