Involvement of epidermal growth factor-like domain of NELL proteins in the novel protein-protein interaction with protein kinase C

Dihydropteroate synthase (DHPS) catalyses a key step in the biosynthesis of folic acid and is the target for inhibition by the sulphonamide class of antimicrobial agents. Here we describe a study of the enzymatic mechanism and sulphonamide inhibition of DHPS from the pathogen Streptococcus pneumoniae. Equilibrium binding assays showed that binding of the substrate para-aminobenzoic acid (pABA) to DHPS was absolutely dependent on the presence of pyrophosphate, which acts as an analogue of the second substrate 6-hydroxymethyl-7, 8-dihydropterin pyrophosphate (DHPPP). The product of the reaction, dihydropteroate, was also able to bind to DHPS. Sulphonamides were capable of displacing pABA in a competitive manner, with equilibrium binding constants that were significantly higher than the equivalent Ki values deduced from steady state kinetic measurements. These results indicate that the target for sulphonamide inhibition of S. pneumoniae DHPS is the enzyme-DHPPP binary complex, rather than the apoprotein form of the enzyme.     

Crystal structure of Mycobacterium tuberculosis 7,8-dihydropteroate synthase in complex with pterin monophosphate: new insight into the enzymatic mechanism and sulfa-drug action

The enzyme 7,8-dihydropteroate synthase (DHPS) catalyzes the condensation of para-aminobenzoic acid (pABA) with 6-hydroxymethyl-7, 8-dihydropterin-pyrophosphate to form 7,8-dihydropteroate and pyrophosphate. DHPS is essential for the de novo synthesis of folate in prokaryotes, lower eukaryotes, and in plants, but is absent in mammals. Inhibition of this enzyme's activity by sulfonamide and sulfone drugs depletes the folate pool, resulting in growth inhibition and cell death. Here, we report the 1.7 A resolution crystal structure of the binary complex of 6-hydroxymethylpterin monophosphate (PtP) with DHPS from Mycobacterium tuberculosis (Mtb), a pathogen responsible for the death of millions of human beings each year. Comparison to other DHPS structures reveals that the M. tuberculosis DHPS structure is in a unique conformation in which loop 1 closes over the active site. The Mtb DHPS structure hints at a mechanism in which both loops 1 and 2 play important roles in catalysis by shielding the active site from bulk solvent and allowing pyrophosphoryl transfer to occur. A binding mode for pABA, sulfonamides and sulfones is suggested based on: (i) the new conformation of the closed loop 1; (ii) the distribution of dapsone and sulfonamide resistance mutations; (iii) the observed direction of the bond between the 6-methyl carbon atom and the bridging oxygen atom to the alpha-phosphate group in the Mtb DHPS:PtP binary complex; and (iv) the conformation of loop 2 in the Escherichia coli DHPS structure. Finally, the Mtb DHPS structure reveals a highly conserved pterin binding pocket that may be exploited for the design of novel antimycobacterial agents.     

Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the pathway for folic acid biosynthesis. The enzyme has been identified as a potential target for antimicrobial drugs. Equilibrium binding studies showed that Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue alpha, beta-methyleneadenosine triphosphate (AMPCPP) with high affinity. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (MANT-ATP) exhibited a substantial fluorescence enhancement upon binding to HPPK, with an equilibrium dissociation constant comparable with that for ATP (10.4 and 4.5 micrometer, respectively). The apoenzyme did not bind the second substrate HMDP, however, unless AMPCPP was present, suggesting that the enzyme binds ATP first, followed by HMDP. Equilibrium titration of HPPK into HMDP and AMPCPP showed an enhancement of fluorescence from the pterin ring of the substrate, and a dissociation constant of 36 nm was deduced for HMDP binding to the HPPK.AMPCPP binary complex. Stopped flow fluorimetry measurements showed that the rate constants for the binding of MANT-ATP and AMPCPP to HPPK were relatively slow (3.9 x 10(5) and 1.05 x 10(5) m(-1) s(-1), respectively) compared with the on rate for binding of HMDP to the HPPK.AMPCPP binary complex. The significance of these results with respect to the crystal structures of HPPK is discussed.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase

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.     

Crystal structure of 7,8-dihydropteroate synthase from Bacillus anthracis: mechanism and novel inhibitor design

Dihydropterate synthase (DHPS) is the target for the sulfonamide class of antibiotics, but increasing resistance has encouraged the development of new therapeutic agents against this enzyme. One approach is to identify molecules that occupy the pterin binding pocket which is distinct from the pABA binding pocket that binds sulfonamides. Toward this goal, we present five crystal structures of DHPS from Bacillus anthracis, a well-documented bioterrorism agent. Three DHPS structures are already known, but our B. anthracis structures provide new insights into the enzyme mechanism. We show how an arginine side chain mimics the pterin ring in binding within the pterin binding pocket. The structures of two substrate analog complexes and the first structure of a DHPS-product complex offer new insights into the catalytic mechanism and the architecture of the pABA binding pocket. Finally, as an initial step in the development of pterin-based inhibitors, we present the structure of DHPS complexed with 5-nitro-6-methylamino-isocytosine.     

Biochemical and structural characterization of the putative dihydropteroate synthase ortholog Rv1207 of Mycobacterium 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 (H(2)PtPP). 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 H(2)PtPP 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 H(2)PtPP-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.     

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae

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.     

Crystal structure of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase•dihydropteroate synthase bifunctional enzyme from Francisella tularensis

The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases.     

Cytosolic hydroxymethyldihydropterin pyrophosphokinase/dihydropteroate synthase from Arabidopsis thaliana: a specific role in early development and stress response

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.     

Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme

Folate derivatives are essential cofactors in the biosynthesis of purines, pyrimidines and amino acids across all forms of life. Mammals uptake folate from their diets, whereas most bacteria must synthesize folate de novo. Therefore, the enzymes in the folate biosynthetic pathway are attractive drug targets against bacterial pathogens such as Mycobacterium tuberculosis, the cause of the world's most deadly infectious disease, tuberculosis (TB). M.tuberculosis 7,8-dihydroneopterin aldolase (Mtb FolB, DHNA) is the second enzyme in the folate biosynthetic pathway, which catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and glycoaldehyde. The 1.6A X-ray crystal structure of Mtb FolB complexed with its product, 6-hydroxymethyl-7,8-dihydropterin, reveals an octameric assembly similar to that seen in crystal structures of other FolB homologs. However, the 2.5A crystal structure of unliganded Mtb FolB reveals a novel tetrameric oligomerization state, with only partially formed active sites. A substrate induced conformational change appears to be necessary to convert the inactive tetramer to the active octamer. Ultracentrifugation confirmed that in solution unliganded Mtb FolB is mainly tetrameric and upon addition of substrate FolB is predominantly octameric. Kinetic analysis of substrate binding gives a Hill coefficient of 2.0, indicating positive cooperativity. We hypothesize that Mtb FolB displays cooperativity in substrate binding to regulate the cellular concentration of 7,8-dihydroneopterin, so that it may function not only as a precursor to folate but also as an antioxidant for the survival of M.tuberculosis against host defenses.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: A novel assay for dihydropteroate synthase

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 MgPP_i to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/α-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.     

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K(d) and IC(50) values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.     

Catalytic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: site-directed mutagenesis and biochemical studies

The roles of a pair of conserved positively charged residues R82 and R92 at a catalytic loop of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) have been investigated by site-directed mutagenesis and biochemical analysis. In the structure of HPPK in complex with ATP and a 6-hydroxymethyl-7,8-dihydropterin (HP) analogue, the guanidinium group of R82 forms two hydrogen bonds with the alpha-phosphate and that of R92 two hydrogen bonds with the beta-phosphate. In the structure of HPPK in complex with alpha,beta-methyleneadenosine triphosphate (AMPCPP, an ATP analogue) and HP, the guanidinium group of R82 has no direct interaction with AMPCPP and that of R92 forms two hydrogen bonds with the alpha-phosphate. Substitution of R82 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 380, but there were no significant changes in the binding energy or binding kinetics of either substrate. Substitution of R92 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 3.5 x 10(4). The mutation caused no significant changes in the binding energy or binding kinetics of MgATP. It did not cause a significant change in the binding energy of HP either but caused a decrease in the association rate constant for the binding of HP by a factor of approximately 4.5 and a decrease in the dissociation rate constant by a factor of approximately 10. The overall structures of the ternary complexes of both mutants were very similar to the corresponding structure of wild-type HPPK as described in the companion paper. The results suggest that R82 does not contribute to the binding of either substrate, and R92 is dispensable for the binding of MgATP but plays a role in facilitating the binding of HP. Both R82 and R92 are important for catalysis, and R92 plays a critical role in the transition state stabilization.     

Reaction trajectory of pyrophosphoryl transfer catalyzed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two products. HPPK is a key enzyme in the folate biosynthetic pathway and is essential for microorganisms but absent from mammals. For the HPPK-catalyzed pyrophosphoryl transfer, a reaction coordinate is constructed on the basis of the thermodynamic and transient kinetic data we reported previously, and the reaction trajectory is mapped out with five three-dimensional structures of the enzyme at various liganded states. The five structures are apo-HPPK (ligand-free enzyme), HPPK.MgATP(analog) (binary complex of HPPK with its first substrate) and HPPK.MgATP(analog).HP (ternary complex of HPPK with both substrates), which we reported previously, and HPPK.AMP.HPPP (ternary complex of HPPK with both product molecules) and HPPK.HPPP (binary complex of HPPK with one product), which we present in this study.     

Essential roles of a dynamic loop in the catalysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphoryl group from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP) following an ordered bi-bi mechanism with ATP as the first substrate. The rate-limiting step of the reaction is product release, and the complete active center is assembled and sealed only upon the binding of both ATP and HP. The assembly of the active center involves large conformational changes in three catalytic loops, among which loop 3 undergoes the most dramatic and unusual changes. To investigate the roles of loop 3 in catalysis, we have made a deletion mutant, which has been investigated by biochemical and X-ray crystallographic analysis. The biochemical data showed that the deletion mutation does not have significant effects on the dissociation constants or the rate constants for the binding of the first substrate MgATP or its analogues. The dissociation constant of HP for the mutant increases by a factor of approximately 100, which is due to a large increase in the dissociation rate constant. The deletion mutation causes a shift of the rate-limiting step in the reaction and a decrease in the rate constant for the chemical step by a factor of approximately 1.1 x 10(5). The crystal structures revealed that the deletion mutation does not affect protein folding, but the catalytic center of the mutant is not fully assembled even upon the formation of the ternary complex and is not properly sealed. The results together suggest that loop 3 is dispensable for the folding of the protein and the binding of the first substrate MgATP, but is required for the assembling and sealing of the active center. The loop plays an important role in the stabilization of the ternary complex and is critical for catalysis.     

Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg(2+) is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is approximately 50 times larger than that for the steady-state phase. The latter is very similar to the k(cat) value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.     

An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites

Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.     

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K_d, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC₅₀ of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and ¹⁵N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including ¹⁵N-¹H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.     

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 [(3)H]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 2.3A. Structural information from these crystals could be used to design novel drugs to inhibit folate biosynthesis.