Dihydropteroate Synthase Activity Research Articles

Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisellatularensis.

Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisellatularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. Although Pemble etal. (PLoS One 5, e14165) determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme. In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity, and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 of that of Escherichiacoli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing therapeutic agents for tularemia, and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of the genomes of ten F.tularensis strains indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F.tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F.tularensis. The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.

Fragment-Based Approach Identifies a Novel Inhibitory Site on DHPS

Dihydropteroate synthase (DHPS) is an essential enzyme in the bacterial folate biosynthetic pathway. It catalyzes the condensation of 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPP) with p-aminobenzoic acid (pABA) to form the folate intermediate, 7,8-dihydropteroate. DHPS is the target of the sulfonamide class of antibiotics. Widespread resistance to sulfonamides has decreased their clinical use. The active site of DHPS is comprised of three sub-sites: the structured “pterin” site, the flexible pABA site, and the anion binding pocket. Most of the drug resistant mutations have been mapped to the pABA sub-site of DHPS. using an NMR ligand-based screening approach, a number of structurally unrelated fragment-like small molecules have been identified that inhibit the enzymatic activity of DHPS from Bacillus anthracis (Ba), Yersinia pestis (Yp), and Staphylococcus aureus (Sa). Fragment hits were shown to target the three sub-pockets of the active site and a novel site distinct from the active site. The latter site potentially inhibits via an allosteric mechanism and has been characterized by high resolution X-ray crystallography.We screened the Maybridge® Fragment library of 1,100 fragments using water ligand observed gradient spectroscopy (waterLOGSY) as a primary screen which resulted in a hit rate of 6.7 %. Of the 74 hits, 25 were shown to inhibit DHPS activity using two independent enzyme activity assays. A total of eight compounds inhibited the activity of DHPS from three different species (Ba, Yp, and Sa). In addition to screening for inhibition, the fragment hits were validated using a number of biophysical techniques including 2D NMR, SPR, competition waterLOGSY, and X-ray crystallography. Herein, we focus on two fragment hits for which high-resolution x-ray crystal structures are available

Biochemical and structural characterization of the putative dihydropteroate synthase ortholog Rv1207 ofMycobacterium tuberculosis

Dihydropteroate synthase (DHPS) is involved in de novo biosynthesis of the essential cofactor folate by catalyzing the condensation of para-aminobenzoic acid (pABA) and 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (H2PtPP). Mycobacterium tuberculosis possesses a functional DHPS (MtDHPS, Rv3608c, folP1) and, based on sequence similarities, a putative ortholog (Rv1207, folP2). Here, we demonstrate that Rv1207 shows a low H2PtPP substrate affinity and lacks enzymatic DHPS activity. However, we found dapsone, a structural analog of pABA and clinically used DHPS inhibitor, to weakly bind both proteins. To gain insights into the lack of DHPS activity of Rv1207, its three-dimensional structure was determined at 2.64 A. The overall fold of both, MtDHPS (1EYE) and Rv1207, is highly conserved and conforms to a classical triosephosphate isomerase barrel arrangement. The predicted H2PtPP-binding pocket of Rv1207 is occupied by a histidine side chain, relative to a leucine residue in MtDHPS, consistent with the low affinity for this substrate and the lack of DHPS activity. We conclude that folP2 does not encode a DHPS and therefore cannot act as bypass for folP1. The metabolic function of Rv1207 remains to be defined.

Purification, properties, and crystallization of Saccharomyces cerevisiae dihydropterin pyrophosphokinase-dihydropteroate synthase

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 His 6 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 Å. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.

Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli

Pneumocystis pneumonia or PCP is caused by Pneumocystis jirovecii, an obligate parasite of the human lung. In this study P. jirovecii genomic sequence encoding FAS, a trifunctional protein including dihydroneopterin aldolase (DHNA), hydroxymethyldihydropterin pyrophosphokinase (PPPK) and dihydropteroate synthase (DHPS) were identified by PCR amplification from fixed broncheolar lavage samples from patients having Pneumocystis pneumonia. The P. jirovecii trifunctional DHNA–PPPK–DHPS genes (PjFAS) showed a high degree of conservation with the rat Pneumocystis carinii and P. carinii f. sp. macaca sequences. To test the functionality of the PjFAS sequences introns were removed followed by cloning and expression of PjFAS sequences in a DHPS-disrupted Escherichia coli strain. Complementation depended on the presence of N-terminal FAS sequences in addition to a glutathione S- transferase tag to the N-terminus of PjFAS. Functional complementation allowed evaluation of DHPS mutations implicated with sulfa drug resistance.

Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human malaria parasite Plasmodium falciparum

Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosynthetic pathway converts GTP to polyglutamated derivatives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfection system adapted to studying this pathway, we investigated modulation of dihydropteroate synthase (DHPS) activity on parasite phenotypes. Dihydropteroate synthase incorporates p-aminobenzoate (pABA) into dihydropteroate, the precursor of dihydrofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full-length but mutated at two key residues and having < 10% of normal activity were viable in folate-supplemented medium. Metabolic labelling showed that these parasites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfadoxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo. However, we found no evidence of a significant role for DHPS in folate salvage. Moreover, when biosynthesis was compromised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.

Arabidopsis dihydropteroate synthase: General properties and inhibition by reaction product and sulfonamides

Dihydropteroate synthase (DHPS) (EC 2.5.1.15) was extracted from leaves of Arabidopsis thaliana and purified 21-fold by ion-exchange chromatography. This enzyme preparation was then characterized for several of its properties. Michaelis-Menten constants for the substrates, p-aminobenzoic acid and dihydropteridine diphosphate were estimated to be 2.5 and 91 μM, respectively. In an optimized assay, the reaction product, dihydropteroic acid, competitively inhibited the enzyme activity with a K i of 81 μM. However, neither dihydrofolate nor tetrahydrofolate, products further downstream in the biosynthetic pathway inhibited enzymatic activity. This appears to be the first report of product inhibition of DHPS from a higher plant. The relative inhibitory properties of several sulfonamides, analogues of p-aminobenzoic acid, were also examined. The substitutions on the amide nitrogen of the sulfonamides influenced the degree of inhibition; thus I 50 values for the inhibition of the DHPS activity by sulfanilamide, sulfacetamide and sulfadiazine were estimated to be 18.6, 9.6 and 4.2 μM, respectively. The competitive pattern of inhibition was shown in experiments with sulfadiazine.