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Ian G. Macreadie - One of the best experts on this subject based on the ideXlab platform.
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Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase.
Protein Expression and Purification, 2005Co-Authors: Janette Berglez, Patricia A. Pilling, Ian G. Macreadie, Ross T. FernleyAbstract:Abstract The tri-functional enzyme of Saccharomyces cerevisiae dihydroneopterin aldolase (DHNA)—dihydropterin pyrophosphokinase (PPPK)—dihydropteroate synthase (DHPS) catalyzes three sequential steps in folate biosynthesis. A cDNA encoding the PPPK and DHPS domains of the tri-functional enzyme has been cloned. This bi-functional enzyme was expressed as a His6 fusion protein in Escherichia coli and the protein was purified to apparent homogeneity. The purified protein possesses both PPPK and DHPS activities as measured by the incorporation of [3H]p-ABA into the appropriate substrate. The pH optimum of the DHPS activity was determined to be 8.5. Gel filtration measurement indicates that the protein exists as a dimer in solution. A robotic screening method was used to identify crystallization conditions. Bi-pyramidal crystals of the enzyme formed with the protein in the presence of a pterin substrate analog in phosphate buffer (pH 6.3) and these diffracted to 2.3 A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.
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the three dimensional structure of the bifunctional 6 hydroxymethyl 7 8 dihydropterin pyrophosphokinase dihydropteroate synthase of saccharomyces cerevisiae
Journal of Molecular Biology, 2005Co-Authors: Michael C Lawrence, Janette Berglez, Patricia A. Pilling, Ross T. Fernley, Peter Iliades, Ian G. MacreadieAbstract:In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species.
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dihydropteroate synthase mutations in pneumocystis jiroveci can affect sulfamethoxazole resistance in a saccharomyces cerevisiae model
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Peter Iliades, Steven R Meshnick, Ian G. MacreadieAbstract:Dihydropteroate synthase (DHPS) mutations in Pneumocystis jiroveci have been associated epidemiologically with resistance to sulfamethoxazole (SMX). Since P. jiroveci cannot be cultured, inherent drug resistance cannot be measured. This study explores the effects of these mutations in a tractable model organism, Saccharomyces cerevisiae. Based on the sequence conservation between the DHPS enzymes of P. jiroveci and S. cerevisiae, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in P. jiroveci were reverse engineered into the S. cerevisiae DHPS. Those mutations, T597A and P599S, can occur singly but are most commonly found together and are associated with SMX treatment failure. Mutations encoding the corresponding changes in the S. cerevisiae DHPS were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase. A yeast strain with DHPS deleted was employed as the host strain, and transformants having DHPS activity were recovered. Mutants having both T597 and P599 substitutions had a requirement for p-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding. These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance. Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions.
Joseph A. Kovacs - One of the best experts on this subject based on the ideXlab platform.
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mutations in the dihydropteroate synthase gene of human derived pneumocystis carinii isolates from italy are infrequent but correlate with prior sulfa prophylaxis
The Journal of Infectious Diseases, 2002Co-Authors: Joseph A. Kovacs, Antonella Valerio, Giovanna Fantoni, Antonietta Cargnel, Chiara AtzoriAbstract:Mutations in the human-derived Pneumocystis carinii dihydropteroate synthase (DHPS) gene have been reported with increasing frequency and have been linked to prior sulfa prophylaxis and possible emergence of sulfa resistance. This study was done to examine the prevalence and clinical significance of P. carinii DHPS mutations in Italian patients. A previously described singlestrand conformation polymorphism technique was used to identify P. carinii DHPS mutations in 107 patients with acquired immunodeficiency syndrome. Overall prevalence (8%) was low compared with that in other reports. Mutations were observed in 19% (6/31) of patients exposed to sulfa prophylaxis, compared with 4% (3/76) of patients not exposed to sulfa prophylaxis (P = .017). No significant association was observed between the presence of DHPS mutations and mortality, CD4 cell count, or demographic factors. The study confirms the association between DHPS mutations and prior sulfa prophylaxis and shows that the prevalence of DHPS mutations in an Italian patient population is lower than that in other populations.
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pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim sulfamethoxazole or dapsone use
The Journal of Infectious Diseases, 1999Co-Authors: Luciana Borio, Henry Masur, Joseph A. KovacsAbstract:Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations ( ). In addition to suggesting that there is less selective P ! .001
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pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim sulfamethoxazole or dapsone use
The Journal of Infectious Diseases, 1999Co-Authors: Luciana Borio, Henry Masur, Joseph A. KovacsAbstract:Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations (P<.001). In addition to suggesting that there is less selective pressure on DHFR than on DHPS, this study reinforces the hypothesis that mutations in the DHPS gene are likely involved in the development of sulfa resistance in P. carinii.
Laurence Huang - One of the best experts on this subject based on the ideXlab platform.
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high prevalence of pneumocystis jirovecii dihydropteroate synthase gene mutations in patients with a first episode of pneumocystis pneumonia in santiago chile and clinical response to trimethoprim sulfamethoxazole therapy
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Carolina A Ponce, Olga Matos, Laurence Huang, Magali Chabe, Claudio George, Alejandra Cardenas, Luisa Duran, Julia Guerrero, Rebeca Bustamante, R F MillerAbstract:Mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis jirovecii are associated with the failure of sulfa prophylaxis. They can develop by selection in patients receiving sulfa drugs or be acquired via person-to-person transmission. DHPS mutations raise concern about the decreasing efficacy of sulfa drugs, the main available therapeutic tool for Pneumocystis pneumonia (PCP). The prevalence of Pneumocystis DHPS mutations was examined in Pneumocystis isolates from 56 sulfa-prophylaxis-naive adults with a first episode of PCP from 2002 to 2010 in Santiago, Chile. Their clinical history was reviewed to analyze the effect of these mutations on response to trimethoprim-sulfamethoxazole (TMP-SMX) therapy and outcome. Mutant genotypes occurred in 22 (48%) of 46 HIV-infected patients and in 5 (50%) of 10 HIV-uninfected patients. Compared to patients with a wild-type genotype, those with mutant genotypes were more likely to experience sulfa treatment-limiting adverse reactions and to have a twice-longer duration of mechanical ventilation if mechanically ventilated. Specific genotypes did not associate with death, which occurred in none of the HIV-infected patients and in 50% of the non-HIV-infected patients. Chile has a high prevalence of DHPS mutations, which were presumably acquired through interhuman transmission because patients were not on sulfa prophylaxis. These results contrast with the low prevalence observed in other Latin American countries with similar usage of sulfa drugs, suggesting that additional sources of resistant genotypes may be possible. The twice-longer duration of mechanical ventilation in patients with mutant DHPS genotypes suggests a decreased efficacy of TMP-SMX and warrants collaborative studies to assess the relevance of DHPS mutations and further research to increase therapeutic options for PCP.
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severity and outcome of hiv associated pneumocystis pneumonia containing pneumocystis jirovecii dihydropteroate synthase gene mutations
AIDS, 2005Co-Authors: Kristina Crothers, Charles B Beard, Joan Turner, Gena Groner, Melissa Fox, Alison Morris, Shary Eiser, Laurence HuangAbstract:Background The impact of Pneumocystis jirovecii (formerly P. carinii) dihydropteroate synthase (DHPS) gene mutations on morbidity and mortality of Pneumocystis pneumonia (PCP) in HIV-positive patients is unclear. Objective To determine whether severity and outcome of HIV-associated PCP differs according to DHPS genotype. Setting A prospective, observational study in a university-affiliated county hospital. Patients The study included 197 patients with 215 microscopically confirmed PCP episodes and successfully sequenced DHPS genotypes; 175 (81%) episodes displayed mutant genotypes. Main outcome measure All-cause mortality within 60 days. Results The majority of patients (86%) with PCP containing Pneumocystis DHPS mutations survived. Although severity of PCP was comparable, there was a trend for more patients with mutant genotypes than patients with wild-type to require mechanical ventilation (14.3% versus 2.5%; P = 0.056) and to die (14.3% versus 7.5%, P = 0.31). Independent predictors of mortality at baseline were low serum albumin levels [odds ratio (OR), 4.62; 95% confidence interval (CI), 1.63-13.1; P = 0.004] and requiring intensive care within 72 h of hospitalization (OR, 5.06; 95% CI, 1.43-18.0; P = 0.012). Conclusion The majority of HIV-infected patients with PCP containing mutant Pneumocystis DHPS genotypes survived. Mortality was related primarily to the underlying severity of illness. However, a trend towards increased mortality in episodes of PCP containing mutant DHPS genotypes was observed and this warrants further study.
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effect of mutations in pneumocystis carinii dihydropteroate synthase gene on outcome of p carinii pneumonia in patients with hiv 1 a prospective study
The Lancet, 2001Co-Authors: Thomas R Navin, Charles B Beard, Laurence Huang, Carlos Del Rio, Sherline Lee, Norman J Pieniazek, Jane L Carter, Allen W Hightower, David RimlandAbstract:Summary Background Investigators have reported that patients infected with Pneumocystis carinii containing mutations in the DHPS (dihydropteroate synthase) gene have a worse outcome than those infected with P carinii containing wild-type DHPS . We investigated patients with HIV-1 infection and P carinii pneumonia to determine if DHPS mutations were associated with poor outcomes in these patients. Methods We compared presence of mutations at the DHPS locus with survival and response of patients to co-trimoxazole or other drugs. Findings For patients initially given co-trimoxazole, nine (14%) of 66 with DHPS mutant died, compared with nine (25%) of 36 with wild type (risk ratio=0·55 [95% CI=0·24–1·25]; p=0·15). Ten (15%) of 66 patients with a DHPS mutant did not respond to treatment, compared with 13 (36%) of 36 patients with the wild type (0·42 [0·20–0·86]; p=0·02). For patients aged 40 years or older, four (14%) of 29 with the mutant and nine (56%) of 16 with the wild type died (0·25 [0·09–0·67]; p=0·005). Interpretation These results, by contrast with those of previous studies, suggest that patients with wild-type P carinii do not have a better outcome than patients with the mutant when given co-trimoxazole. Our results suggest that presence of a DHPS mutation should be only one of several criteria guiding the choice of initial drug treatment of P carinii pneumonia in patients with HIV-1 infection.
Luciana Borio - One of the best experts on this subject based on the ideXlab platform.
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pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim sulfamethoxazole or dapsone use
The Journal of Infectious Diseases, 1999Co-Authors: Luciana Borio, Henry Masur, Joseph A. KovacsAbstract:Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations ( ). In addition to suggesting that there is less selective P ! .001
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pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim sulfamethoxazole or dapsone use
The Journal of Infectious Diseases, 1999Co-Authors: Luciana Borio, Henry Masur, Joseph A. KovacsAbstract:Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations (P<.001). In addition to suggesting that there is less selective pressure on DHFR than on DHPS, this study reinforces the hypothesis that mutations in the DHPS gene are likely involved in the development of sulfa resistance in P. carinii.
Jyoti Chhibbergoel - One of the best experts on this subject based on the ideXlab platform.
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structure of 6 hydroxymethyl 7 8 dihydropterin pyrophosphokinase dihydropteroate synthase from plasmodium vivax sheds light on drug resistance
Journal of Biological Chemistry, 2018Co-Authors: M Yogavel, Joanne E Nettleship, Akansha Sharma, Karl Harlos, Abhishek Jamwal, Rini Chaturvedi, Manmohan Sharma, V Jain, Jyoti ChhibbergoelAbstract:The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.