Catalysis and sulfa drug resistance in dihydropteroate synthase.
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ABSTRACT:
SUBMITTER: Yun MK
PROVIDER: S-EPMC3531234 | biostudies-literature | 2012 Mar
REPOSITORIES: biostudies-literature
Publications
Catalysis and sulfa drug resistance in dihydropteroate synthase.
Yun Mi-Kyung MK Wu Yinan Y Li Zhenmei Z Zhao Ying Y Waddell M Brett MB Ferreira Antonio M AM Lee Richard E RE Bashford Donald D White Stephen W SW
Science (New York, N.Y.) 20120301 6072
The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results supp ...[more]
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Project description:Staphylococcal species are a leading cause of bacterial drug-resistant infections and associated mortality. One strategy to combat bacterial drug resistance is to revisit compromised targets, and to circumvent resistance mechanisms using structure-assisted drug discovery. The folate pathway is an ideal candidate for this approach. Antifolates target an essential metabolic pathway, and the necessary detailed structural information is now available for most enzymes in this pathway. Dihydropteroate synthase (DHPS) is the target of the sulfonamide class of drugs, and its well characterized mechanism facilitates detailed analyses of how drug resistance has evolved. Here, we surveyed clinical genetic sequencing data in S. aureus to distinguish natural amino acid variations in DHPS from those that are associated with sulfonamide resistance. Five mutations were identified, F17L, S18L, T51M, E208K, and KE257_dup. Their contribution to resistance and their cost to the catalytic properties of DHPS were evaluated using a combination of biochemical, biophysical and microbiological susceptibility studies. These studies show that F17L, S18L, and T51M directly lead to sulfonamide resistance while unexpectedly increasing susceptibility to trimethoprim, which targets the downstream enzyme dihydrofolate reductase. The secondary mutations E208K and KE257_dup restore trimethoprim susceptibility closer to wild-type levels while further increasing sulfonamide resistance. Structural studies reveal that these mutations appear to selectively disfavor the binding of the sulfonamides by sterically blocking an outer ring moiety that is not present in the substrate. This emphasizes that new inhibitors must be designed that strictly stay within the substrate volume in the context of the transition state.
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Project description:The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting dihydropteroate synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.
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Project description:Sulfadoxine-pyrimethamine (SP) combination drug is currently being used in India for the treatment of Plasmodium falciparum as partner drug in artemisinin-based combination therapy (ACT). Resistance to sulfadoxine and pyrimethamine in P. falciparum is linked with mutations in dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes respectively. This study was undertaken to estimate the prevalence of such mutations in pfdhfr and pfdhps genes in four states of India.Plasmodium falciparum isolates were collected from two states of India with high malaria incidence i.e., Jharkhand and Odisha and two states with low malaria incidence i.e., Andhra Pradesh and Uttar Pradesh between years 2006 to 2012. Part of sulfadoxine-pyrimethamine (SP) drug resistance genes, pfdhfr and pfdhps were PCR-amplified, sequenced and analyzed.A total of 217 confirmed P. falciparum isolates were sequenced for both Pfdhfr and pfdhps gene. Two pfdhfr mutations 59R and 108N were most common mutations prevalent in all localities in 77 % of isolates. Additionally, I164L was found in Odisha and Jharkhand only (4/70 and 8/84, respectively). Another mutation 51I was found in Odisha only (3/70). The pfdhps mutations 436A, 437G, 540E and 581G were found in Jharkhand and Odisha only in 13, 26, 14 and 13 % isolates respectively, and was absent in Uttar Pradesh and Andhra Pradesh. Combined together for pfdhps and pfdhfr locus, triple, quadruple, quintuple and sextuple mutations were present in Jharkhand and Odisha while absent in Uttar Pradesh and Andhra Pradesh.While only double mutants of pfdhfr was present in low transmission area (Uttar Pradesh and Andhra Pradesh) with total absence of pfdhps mutants, up to sextuple mutations were present in high transmission areas (Odisha and Jharkhand) for both the genes combined. Presence of multiple mutations in pfdhfr and pfdhps genes linked to SP resistance in high transmission area may lead to fixation of multiple mutations in presence of high drug pressure and high recombination rate.
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Project description:BackgroundSulphadoxine-pyrimethamine (SP), an antifolate, was replaced by artemether-lumefantrine as the first-line malaria drug treatment in Kenya in 2004 due to the wide spread of resistance. However, SP still remains the recommended drug for intermittent preventive treatment in pregnant women and infants (IPTP/I) owing to its safety profile. This study assessed the prevalence of mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes associated with SP resistance in samples collected in Kenya between 2008 and 2012.MethodsField isolates collected from Kisumu, Kisii, Kericho and Malindi district hospitals were assessed for genetic polymorphism at various loci within Pfdhfr and Pfdhps genes by sequencing.ResultsAmong the Pfdhfr mutations, codons N51I, C59R, S108N showed highest prevalence in all the field sites at 95.5%, 84.1% and 98.6% respectively. Pfdhfr S108N prevalence was highest in Kisii at 100%. A temporal trend analysis showed steady prevalence of mutations over time except for codon Pfdhps 581 which showed an increase in mixed genotypes. Triple Pfdhfr N51I/C59R/S108N and double Pfdhps A437G/ K540E had high prevalence rates of 86.6% and 87.9% respectively. The Pfdhfr/Pfdhps quintuple, N51I/C59R/S108N/A437G/K540E mutant which has been shown to be the most clinically relevant marker for SP resistance was observed in 75.7% of the samples.ConclusionSP resistance is still persistently high in western Kenya, which is likely due to fixation of key mutations in the Pfdhfr and Pfdhps genes as well as drug pressure from other antifolate drugs being used for the treatment of malaria and other infections. In addition, there is emergence and increasing prevalence of new mutations in Kenyan parasite population. Since SP is used for IPTP/I, molecular surveillance and in vitro susceptibility assays must be sustained to provide information on the emergence and spread of SP resistance.
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Project description:Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.
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Project description:The characterization of the genetic basis of sulfonamide resistance in Campylobacter jejuni was attempted. The resistance determinant from a sulfonamide-resistant strain of C. jejuni was cloned and was found to show 42% identity with the folP gene (which codes for dihydropteroate synthase, the target of sulfonamides) of the related bacterium Helicobacter pylori. The sequences of the areas surrounding the folP gene in C. jejuni showed similarity to those of the areas surrounding the corresponding gene in H. pylori. The folP gene of C. jejuni, which mediates the resistance, was observed to show particular features when it was compared to other known folP genes. One of these features is the presence of two pairs of direct repeats (15 and 27 bp) within the coding sequence of the gene. Comparison of the C. jejuni folP genes that mediate susceptibility and resistance revealed the occurrence of mutations that changed four amino acid residues. Resistance of C. jejuni to sulfonamides could be associated with one or several of these four mutational substitutions, which all occurred in the five different resistant isolates studied. The codon for one of these changed amino acids was found to be located in the second direct repeat within the coding sequence of the gene. The change made the repeat perfect. The transformation of both the resistance and the susceptibility variants of the gene into an Escherichia coli folP knockout mutant was found to complement the dihydropteroate synthase deficiency, confirming that the characterized sulfonamide resistance determinant codes for the C. jejuni dihydropteroate synthase enzyme. Kinetic measurements established different affinities of sulfonamide for the dihydropteroate synthase enzyme isolated from the resistant and susceptible strains. In conclusion, sulfonamide resistance in C. jejuni was shown to be associated with mutational changes in the chromosomally located gene for dihydropteroate synthase, the target of sulfonamides.
| S-EPMC89439 | biostudies-literature
Project description:Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.
| S-EPMC2804029 | biostudies-literature
Project description:BackgroundThe enzyme dihydropteroate synthase (DHPS) participates in the de novo synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of p-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium Burkholderia cenocepacia is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against Burkholderia and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery.ResultsAn efficient recombinant protein expression system for DHPS from B. cenocepacia (BcDHPS) was prepared, the dimeric enzyme purified in high yield and crystallized. The structure of the apo-enzyme and the complex with the product 7,8-dihydropteroate have been determined to 2.35 Å and 1.95 Å resolution respectively in distinct orthorhombic crystal forms. The latter represents the first crystal structure of the DHPS-pterin product complex, reveals key interactions involved in ligand binding, and reinforces data generated by other structural studies. Comparisons with orthologues identify plasticity near the substrate-binding pocket and in particular a range of loop conformations that contribute to the architecture of the DHPS active site. These structural data provide a foundation for hit discovery. An intriguing observation, an artifact of the analysis, that of a potential sulfenamide bond within the ligand complex structure is mentioned.ConclusionStructural similarities between BcDHPS and orthologues from other Gram-negative species are evident as expected on the basis of a high level of sequence identity. The presence of 7,8-dihydropteroate in the binding site provides details about ligand recognition by the enzyme and the different states of the enzyme allow us to visualize distinct conformational states of loops adjacent to the active site. Improved drugs to combat infections by Burkholderia sp. and related Gram-negative bacteria are sought and our study now provides templates to assist that process and allow us to discuss new ways of inhibiting DHPS.
| S-EPMC3098144 | biostudies-literature