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6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but is absent in mammals. Yet, it is not the target of any existing antibiotics. Hence, this enzyme is an attractive target for developing novel antimicrobial agents. A wealth of structural and mechanistic information has provided solid basis for structure-based design of HPPK inhibitors. Our bisubstrate inhibitors were initially created by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups (HP n A, n = 2, 3, or 4), among which HP 4 A exhibited the highest binding affinity (K d  = 0.47 ± 0.04 μM). Further development was carried out based on high-resolution structures of HPPK in complex with HP 4 A. Replacing the phosphate bridge with a piperidine linked thioether eliminated multiple negative charges of the bridge. Substituting the pterin moiety with 7,7-dimethyl-7,8-dihydropterin improved the binding affinity. Arming the piperidine ring with a carboxyl group and oxidizing the thioether further enhanced the potency, resulting in a druglike inhibitor of HPPK (K d  = 0.047 ± 0.007 μM). None of these inhibitors, however, exhibits bacterial cell permeability. It is most likely due to the lack of active folate transporters in bacteria. Replacing the pterin moiety with a 7-deazagaunine moiety, we have obtained a novel bisubstrate inhibitor (HP-101) showing observable cell permeability toward a Gram-positive bacterium. Here, we report the in vitro activity of HP-101 and its structure in complex with HPPK, providing a framework for structure-based further development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Acinetobacter baumannii is an ESKAPE pathogen that causes endocarditis, pneumonia, blood infections, urinary tract infections, and several other illnesses. In addition, it is mainly responsible for nosocomial infection-related mortality. Gram-negative A. baumannii bacterium (AYE Strain) has high MDR and XDR levels. Due to its function in synthesizing purines and amino acids, folic acid is a significant molecule necessary for the growth of bacteria. The metabolic pathway of folate production is therefore a potential therapeutic target to inhibit bacterial growth. In the current study, the three-dimensional model of 6-Hydroxy-methyl dihydropterinpyrophosphokinase (HPPK) was predicted and subsequently processed through a virtual high throughput screening (vHTS) against compounds from Enamine HTSC library, that could bind to its active site. Three lead candidates (Z73322064, Z354558542, and Z906123504) and a control molecule (7,8 dihydro-7,7-dimethyl-6-hydroxymethlypterin; Accession Number: DB02278) were identified using several screening criteria namely estimated binding affinity, estimated inhibition constant, drug-like properties, ADME properties, mode of binding, and interaction patterns of the screened compounds. The physiological behavior of ligand binding on the HPPK enzyme was then studied using molecular dynamics simulations of apo and ligand bound complexes. This study proposed the following three molecules: Z73322064, Z354338542, and Z906123504 as promising lead candidates against the substrate-binding site of the HPPK enzyme from A. baumannii using global, essential dynamics studies along with MM/PBSA based binding free energy analysis.Communicated by Ramaswamy H. Sarma.

The clinical efficacy of sulfa drugs as antimalarials has declined owing to the evolution of resistance in Plasmodium falciparum (Pf) malaria parasites. In order to understand the basis of this resistance and to design more effective antimalarials, we have solved 13 structures of the bifunctional enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK)-dihydropteroate synthase (DHPS) from wild-type (WT) P. falciparum and sulfa-resistant mutants, both as apoenzyme and as complexes with pteroate (PTA) and sulfa derivatives. The structures of these complexes show that PTA, which effectively inhibits both the WT and mutants, stays in active sites without steric constraint. In contrast, parts of the sulfa compounds situated outside of the substrate envelope are in the vicinity of the resistance mutations. Steric conflict between compound and mutant residue along with increased flexibility of loop D2 in the mutants can account for the reduced compound binding affinity to the mutants. Kinetic data show that the mutants have enhanced enzyme activity compared with the WT. These PfDHPS structural insights are critical for the design of novel, substrate envelope-compliant DHPS inhibitors that are less vulnerable to resistance mutations. DATABASES: The data reported in this paper have been deposited in the Protein Data Bank, www.wwpdb.org. PDB ID codes: 6JWQ for apoWT; 6JWR, 6JWS, and 6JWT for PTA complexes of WT, A437G (3D7), and V1/S; 6JWU, 6JWV, and 6JWW for STZ-DHP complexes of WT, 3D7, and V1/S; 6JWX, 6JWY, and 6JWZ for SDX-DHP complexes of WT, 3D7, and W2; 6KCK, 6KCL, and 6KCM for Pterin/pHBA complexes of WT, TN1, and W2., (© 2020 Federation of European Biochemical Societies.)

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines., (© 2018 International Society of Neuropathology.)

Background: The previous studies have demonstrated the reduction of thiamine diphosphate is specific to Alzheimer's disease (AD) and causal factor of brain glucose hypometabolism, which is considered as a neurodegenerative index of AD and closely correlates with the degree of cognitive impairment. The reduction of thiamine diphosphate may contribute to the dysfunction of synapses and neural circuits, finally leading to cognitive decline., Results: To demonstrate this hypothesis, we established abnormalities in the glucose metabolism utilizing thiamine deficiency in vitro and in vivo, and we found dramatically reduced dendrite spine density. We further detected lowered excitatory neurotransmission and impaired hippocampal long-term potentiation, which are induced by TPK RNAi in vitro. Importantly, via treatment with benfotiamine, Aβ induced spines density decrease was considerably ameliorated., Conclusions: These results revealed that thiamine deficiency contributed to synaptic dysfunction which strongly related to AD pathogenesis. Our results provide new insights into pathogenesis of synaptic and neuronal dysfunction in AD.

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) is a monomeric protein with 158 residues, which undergoes large-scale conformational changes between apo, open, and holo states responding to ligand binding for its function. It has been explored widely as an excellent target for potential antibacterial drug development. However, little is known about how conformational dynamics between the native states influences the substrate recognition and the functionality of enzymatic catalysis. Here, we report a coarse-grained triple-basin structure-based model upon ligand binding to describe such multiple-state system by the molecular dynamics simulation. With our model, we have made theoretical predictions that are in good agreement with the experimental measurements. Our results revealed the intrinsic conformational fluctuations between apo and open states without ligand binding. We found that HPPK can switch to the activated holo state upon the ordered binding of the two ligands (ATP and HP). We uncovered the underlying mechanism by which major induced fit and minor population shift pathways coexist upon ligand binding by quantitative flux analysis. Additionally, we pointed out the structural origin for the conformational changes and identified the key residues as well as contact interactions. We further explored the temperature effect on the conformational distributions and pathway weights. It gave strong support that higher temperatures promote population shift, while the induced fit pathway is always the predominant activation route of the HPPK system. These findings will provide significant insights of the mechanisms of the multistate conformational dynamics of HPPK upon ligand binding.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthetic pathway. Comparison of its X-ray and nuclear magnetic resonance structures suggests that the enzyme undergoes significant conformational change upon binding to its substrates, especially in three catalytic loops. Experimental research has shown that, in its binary form, even bound by analogues of MgATP, loops 2 and 3 remain rather flexible; this raises questions about the putative large-scale induced-fit conformational change of the HPPK-MgATP binary complex. In this work, long-time all-atomic molecular dynamics simulations were conducted to investigate the loop dynamics in this complex. Our simulations show that, with loop 3 closed, multiple conformations of loop 2, including the open, semiopen, and closed forms, are all accessible to the binary complex. These results provide valuable structural insights into the details of conformational changes upon 6-hydroxymethyl-7,8-dihydropterin (HP) binding and biological activities of HPPK. Conformational network analysis and principal component analysis related to the loops are also discussed.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 μM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthetic pathway. Comparison of its X-ray and nuclear magnetic resonance structures suggests that the enzyme undergoes significant conformational change upon binding to its substrates, especially in three catalytic loops. Experimental research has shown that even when confined by crystal contacts, loops 2 and 3 remain rather flexible when the enzyme is in its apo form, raising questions about the putative large-scale induced-fit conformational change of HPPK. To investigate the loop dynamics in a crystal-free environment, we performed conventional molecular dynamics simulations of the apo-enzyme at two different temperatures (300 and 350 K). Our simulations show that the crystallographic B-factors considerably underestimate the loop dynamics; multiple conformations of loops 2 and 3, including the open, semi-open, and closed conformations that an enzyme must adopt throughout its catalytic cycle, are all accessible to the apo-enzyme. These results revise our previous view of the functional mechanism of conformational change upon MgATP binding and offer valuable structural insights into the workings of HPPK. In this paper, conformational network analysis and principal component analysis related to the loops are discussed to support the presented conclusions.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents., (Copyright © 2014. Published by Elsevier Ltd.)

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg(2+-)dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N(9) substitution, or tautomerization arising from N(7) substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N(9)-H···Val46 hydrogen bond, combined with the limited space available around the N(9) position. The water-filled pocket under the N(7) position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N(7) (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N(7) position towards the Mg(2+)-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg(2+) centres or Mg(2+)-binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthesis pathway catalyzing the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, is an attractive target for developing novel antimicrobial agents. Previously, we studied the mechanism of HPPK action, synthesized bisubstrate analog inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed a new generation of bisubstrate inhibitors by replacing the phosphate bridge with a piperidine-containing linkage. To further improve linker properties, we have synthesized a new compound, characterized its protein binding/inhibiting properties, and determined its structure in complex with HPPK. Surprisingly, this inhibitor exhibits a new binding mode in that the adenine base is flipped when compared to previously reported structures. Furthermore, the side chain of amino acid residue E77 is involved in protein-inhibitor interaction, forming hydrogen bonds with both 2' and 3' hydroxyl groups of the ribose moiety. Residue E77 is conserved among HPPK sequences, but interacts only indirectly with the bound MgATP via water molecules. Never observed before, the E77-ribose interaction is compatible only with the new inhibitor-binding mode. Therefore, this compound represents a new direction for further development., (Published by Elsevier Ltd.)

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., (Published by Elsevier Ltd.)

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 IC(50) 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 (15)N 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 (15)N-(1)H 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.

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.

Protein conformational dynamics, despite its significant anharmonicity, has been widely explored by normal mode analysis (NMA) based on atomic or coarse-grained potential functions. To account for the anharmonic aspects of protein dynamics, this study proposes, and has performed, an anharmonic NMA (ANMA) based on the C(alpha)-only elastic network models, which assume elastic interactions between pairs of residues whose C(alpha) atoms or heavy atoms are within a cutoff distance. The key step of ANMA is to sample an anharmonic potential function along the directions of eigenvectors of the lowest normal modes to determine the mean-squared fluctuations along these directions. ANMA was evaluated based on the modeling of anisotropic displacement parameters (ADPs) from a list of 83 high-resolution protein crystal structures. Significant improvement was found in the modeling of ADPs by ANMA compared with standard NMA. Further improvement in the modeling of ADPs is attained if the interactions between a protein and its crystalline environment are taken into account. In addition, this study has determined the optimal cutoff distances for ADP modeling based on elastic network models, and these agree well with the peaks of the statistical distributions of distances between C(alpha) atoms or heavy atoms derived from a large set of protein crystal structures., ((c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Plasmodium falciparum bifunctional hydroxymethylpterin pyrophosphokinase-dihydropteroate synthase (pfHPPK-DHPS) is a crucial enzyme in the de novo folate biosynthesis pathway. The crystal structure is not yet available for this enzyme, however, homology model of the enzyme reported previously revealed the presence of parasite-specific insertions. Alignment of pfHPPK-DHPS with HPPK and DHPS sequences from other microorganisms reveals two insertions relative to the corresponding enzyme in other organisms, i.e. HPPK-1 and HPPK-2. The former encompasses amino acid residues 66-162, while the latter covers residues 213-311. In order to investigate the roles of the two insertions, we constructed a number of mutants in which parts of these two insertions were deleted. Characterization of the mutationally altered proteins revealed that deletions of residues 74-80 in the HPPK-1 sequence of the pfHPPK-DHPS, but not that of the monofunctional pfHPPK, decreased the HPPK activity. A longer deletion (residues 74-86) in the HPPK-1 sequence of the bifunctional pfHPPK-DHPS completely inactivated both HPPK and DHPS activities. However, deletion in the HPPK-2 sequence from residues 247-306 did not disrupt the activities of HPPK and DHPS, but the kinetic properties of the recombinant proteins were slightly changed. The importance of HPPK-1 sequence on the catalytic activities of HPPK and DHPS in the bifunctional pfHPPK-DHPS could have implications for development of inhibitors targeting the non-catalytic region of this chemotherapeutically important enzyme.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), which follows an ordered bi-bi kinetic mechanism with ATP binding to the enzyme first. HPPK undergoes dramatic conformational changes during its catalytic cycle as revealed by X-ray crystallography, and the conformational changes are essential for the enzymatic catalysis as shown by site-directed mutagenesis and biochemical and crystallographic analysis of the mutants. However, the dynamic properties of the enzyme have not been measured experimentally. Here, we report a (15)N NMR relaxation study of the dynamic properties of Escherichia coli HPPK from the apo form to the binary substrate complex with MgATP (represented by MgAMPCPP, an ATP analogue) to the Michaelis complex (ternary substrate complex) with MgATP (represented by MgAMPCPP) and HP (represented by 7,7-dimethyl-6-hydroxypterin, an HP analogue). The results show that the binding of the nucleotide to HPPK does not cause major changes in the dynamic properties of the enzyme. Whereas enzymes are often more rigid when bound to the ligand or the substrate, the internal mobility of HPPK is not reduced and is even moderately increased in the binary complex, particularly in the catalytic loops. The internal mobility of the catalytic loops is significantly quenched upon the formation of the ternary complex, but some mobility remains. The enhanced motions in the catalytic loops of the binary substrate complex may be required for the assembling of the ternary complex. On the other hand, some degrees of mobility in the catalytic loops of the ternary complex may be required for the optimal stabilization of the transition state, which may need the instantaneous adjustment and alignment of the side-chain positions of catalytic residues. Such dynamic behaviors may be characteristic of bisubstrate enzymes.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyses the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), and is an essential enzyme in the biosynthesis of folic acid. It is also a potential target for antimicrobial drugs. HPPK from Escherichia coli, which has been the most intensively investigated, is a monomeric protein with a molecular mass of about 18,000. Structures of the enzyme, determined by X-ray crystallography and NMR, have shown that it adopts an alpha/beta fold with a substrate-binding cleft on the surface. Three loop regions surround the enzyme active site and form intimate contacts with the substrates. The enzyme has a fixed order of substrate binding, with ATP binding first, followed by HMDP. Binding of ATP causes a shift in the conformations of the loop regions, which completes formation of the HMDP-binding site. Two magnesium ions bind within the active site, bridging between the phosphate groups in ATP and the enzyme. Both ions appear to play an integral role in ATP recognition and stabilization of the transition state of the reaction. Ligand binding and kinetic studies have shown that the overall rate of the reaction is not limited by the rate of substrate transformation into products on the enzyme, which is relatively fast, but is more likely caused by a slow step associated with product release. These fundamental studies open up the potential for exploitation through the design of specific HPPK inhibitors.

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.

Sampling of small-scale and large-scale motions is important in various computational tasks, such as protein-protein docking and ligand binding. Here, we report further development and applications of the activation-relaxation technique for internal coordinate space trajectories (ARTIST). This method generates conformational moves of any complexity and size by identifying and crossing well-defined saddle points connecting energy minima. Simulations on two all-atom proteins and three protein complexes containing between 70 and 300 amino acids indicate that ARTIST opens the door to the full treatment of all degrees of freedom in dense systems such as protein-protein complexes.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. HPPK undergoes dramatic conformational changes during its catalytic cycle, and the conformational changes are essential for enzymatic catalysis. Thus, the enzyme is not only an attractive target for developing antimicrobial agents but also an excellent model system for studying the catalytic mechanism of enzymatic pyrophosphoryl transfer as well as the role of protein dynamics in enzymatic catalysis. In the present study, we report the NMR solution structures of the binary complex HPPK*MgAMPCPP and the ternary complex HPPK*MgAMPCPP*DMHP, where alpha,beta-methyleneadenosine triphosphate (AMPCPP) and 7, 7-dimethyl-6-hydroxypterin (DMHP) are the analogues of the substrates ATP and HP, respectively. The results suggest that the three catalytic loops of the binary complex of HPPK can assume multiple conformations in slow exchanges as evidenced by multiple sets of NMR signals for several residues in loops 2 and 3 and the very weak or missing NH cross-peaks for several residues in loops 1 and 3. However, the ternary complex shows only one set of NMR signals, and the cross-peak intensities are rather uniform, suggesting that the binding of the second substrate shifts the multiple conformations of the binary complex to an apparently single conformation of the ternary complex. The NMR behaviors and conformations of the binary complex HPPK*MgAMPCPP are significantly different from those of HPPK in complex with Mgbeta,gamma-methyleneadenosine triphosphate (MgAMPPCP). It is suggested that the conformational properties of the binary substrate complex HPPK*MgATP be represented by those of HPPK*MgAMPCPP, because MgAMPCPP is a better MgATP analogue for HPPK with respect to both binding affinity and bound conformation.

The enzymes dihydroneopterin aldolase (DHNA) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyse two consecutive steps in the biosynthesis of folic acid. Neither of these enzymes has a counterpart in mammals, and they have therefore been suggested as ideal targets for antimicrobial drugs. Some of the enzymes within the folate pathway can occur as bi- or trifunctional complexes in bacteria and parasites, but the way in which bifunctional DHNA-HPPK enzymes are assembled is unclear. Here, we report the determination of the structure at 2.9 A resolution of the DHNA-HPPK (SulD) bifunctional enzyme complex from the respiratory pathogen Streptococcus pneumoniae. In the crystal, DHNA is assembled as a core octamer, with 422 point group symmetry, although the enzyme is active as a tetramer in solution. Individual HPPK monomers are arranged at the ends of the DHNA octamer, making relatively few contacts with the DHNA domain, but more extensive interactions with adjacent HPPK domains. As a result, the structure forms an elongated cylinder, with the HPPK domains forming two tetramers at each end. The active sites of both enzymes face outward, and there is no clear channel between them that could be used for channelling substrates. The HPPK-HPPK interface accounts for about one-third of the total area between adjacent monomers in SulD, and has levels of surface complementarity comparable to that of the DHNA-DHNA interfaces. There is no "linker" polypeptide between DHNA and HPPK, reducing the conformational flexibility of the HPPK domain relative to the DHNA domain. The implications for the organisation of bi- and trifunctional enzyme complexes within the folate biosynthesis pathway are discussed.

Biophysical label-free assays such as those based on SPR are essential tools in generating high-quality data on affinity, kinetic, mechanistic and thermodynamic aspects of interactions between target proteins and potential drug candidates. Here we show examples of the integration of SPR with bioinformatic approaches and mutation studies in the early drug discovery process. We call this combination 'structure-based biophysical analysis'. Binding sites are identified on target proteins using information that is either extracted from three-dimensional structural analysis (X-ray crystallography or NMR), or derived from a pharmacore model based on known binders. The binding site information is used for in silico screening of a large substance library (e.g. available chemical directory), providing virtual hits. The three-dimensional structure is also used for the design of mutants where the binding site has been impaired. The wild-type target and the impaired mutant are then immobilized on different spots of the sensor chip and the interactions of compounds with the wild-type and mutant are compared in order to identify selective binders for the binding site of the target protein. This method can be used as a cost-effective alternative to high-throughput screening methods in cases when detailed binding site information is available. Here, we present three examples of how this technique can be applied to provide invaluable data during different phases of the drug discovery process.

Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.

Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyses the transfer of pyrophosphate from ATP to HMDP (6-hydroxymethyl-7,8-dihydropterin), to form AMP and DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). This transformation is a key step in the biosynthesis of folic acid, and HPPK is consequently a target for antimicrobial drugs. The substrates are known to bind to HPPK in an ordered manner, with ATP binding first followed by HMDP. In the present study we show by isothermal titration calorimetry that the product, DHPPP, can bind to the HPPK apoenzyme with high affinity (equilibrium dissociation constant, K(d)=0.2 microM), but without the enhancement of pterin fluorescence that occurs on binding of HMDP. The transient kinetics of the enzyme can be monitored by measuring the change in the fluorescence of the pterin ring using stopped-flow methods. The fluorescence exhibits a pronounced biphasic behaviour: it initially rises and then declines back to its original level. This behaviour is in agreement with a two-state kinetic model, with the first phase of fluorescence increase associated with HMDP binding to the enzyme, and the second phase with a slow event that occurs after the reaction has taken place. The HPPK-DHPPP and HPPK-DHPPP-AMP complexes were examined by NMR, and the binding site for DHPPP partially mapped from changes in chemical shifts identified from two dimensional 1H/15N heteronuclear single-quantum coherence spectra. The results demonstrate that DHPPP, in contrast to HMDP, is able to bind to the HPPK apoenzyme and suggest that the pyrophosphate moieties on the ligand play an important role in establishment of a high affinity binding site for the pterin ring.

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.

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) belongs to a class of catalytic enzymes involved in phosphoryl transfer and is a new target for the development of novel antimicrobial agents. In the present study, the fundamental consideration is to view the overall structure of HPPK as a network of interacting residues and to extract the most cooperative collective motions that define its global dynamics. A coarse-grained model, harmonically constrained according to HPPK's crystal structure is used. Four crystal structures of HPPK (one apo and three holo forms with different nucleotide and pterin analogs) are studied with the goal of providing insights about the function-dynamic correlation and ligand induced conformational changes. The dynamic differences are examined between HPPK's apo- and holo-forms, because they are involved in the catalytic reaction steps. Our results indicate that the palm-like structure of HPPK is nearly rigid, whereas the two flexible loops: L2 (residues 43-53) and L3 (residues 82-92) exhibit the most concerted motions for ligand recognition and presumably, catalysis. These two flexible loops are involved in the recognition of HPPKs nucleotide and pterin ligands, whereas the rigid palm region is associated with binding of these cognate ligands. Six domains of collective motions are identified, comprised of structurally close but not necessarily sequential residues. Two of these domains correspond to the flexible loops (L2 and L3), whereas the remaining domains correspond to the rigid part of the molecule., (Published 2002 Wiley-Liss, Inc.)

The plant enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (HPPK/DHPS) is a mitochondrial bifunctional protein involved in tetrahydrofolate synthesis. The first domain (HPPK) catalyses the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (dihydropterin) by ATP, leading to 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (dihydropterinPP(i)) and AMP. The second domain (DHPS) catalyses the next step, i.e. the condensation of p-aminobenzoic acid (p-ABA) with dihydropterinPP(i) to give 7,8-dihydropteroate (dihydropteroate) and PP(i). In the present article we studied the coupling between these two reactions. Kinetic data obtained for the HPPK domain are consistent with an ordered Bi Bi mechanism where ATP binds first and dihydropterinPP(i) is released last, as proposed previously for the monofunctional Escherichia coli enzyme. In the absence of p-ABA, AMP and dihydropterinPP(i) accumulate and negatively regulate the reaction. In the presence of p-ABA, the rates of AMP and dihydropteroate synthesis are similar, indicating a good coupling between the two reactions. DihydropterinPP(i), an intermediate of the two reactions, never accumulates in this situation. The high specific activity of DHPS relative to HPPK, rather than a preferential channelling of dihydropterinPP(i) between the two catalytic sites, could explain these kinetic data. The maximal velocity of the DHPS domain is limited by the availability of dihydropterinPP(i). It is strongly feedback-inhibited by dihydropteroate and also dihydrofolate and tetrahydrofolate monoglutamate, two intermediates synthesized downstream in the folate biosynthetic pathway. Thus the HPPK domain of this bifunctional protein is the limiting factor of the overall reaction, but the DHPS domain is a potential key regulatory point of the whole folate biosynthetic pathway.

Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in folate pathway, and therefore, is an ideal target for developing novel antimicrobial agents. Because of its small size and high thermal stability, E. coli HPPK is also an excellent model enzyme for studying the mechanisms of enzymatic pyrophosphoryl transfer. We have determined the crystal structures of HPPK in the unligated form and in complex with HP, two Mg2+ ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). Comparison of the two crystal structures reveals dramatic conformational changes of three flexible loops and many side chains and possible roles of the active site residues.

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.

Background: Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coli is a 158-residue thermostable protein that provides a convenient model system for mechanistic studies. Crystal structures have been reported for HPPK without bound ligand, containing an HP analog, and complexed with an HP analog, two Mg(2+) ions, and ATP., Results: We present the 1.25 A crystal structure of HPPK in complex with HP, two Mg(2+) ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveals dramatic conformational changes of three flexible loops and many sidechains. The coordination of Mg(2+) ions has been defined and the roles of 26 residues have been derived., Conclusions: HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.

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.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7, 8-dihydropterin, the first reaction in the folate biosynthetic pathway. Like other enzymes in the folate pathway, HPPK is an ideal target for development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from humans and animals. Using 3'(2')-o-anthraniloyladenosine 5'-triphosphate as a fluorescent probe, a fluorometric competitive binding assay has been developed for measuring the dissociation constants of various compounds that bind to the ATP site of HPPK. The fluorometric assay has been used to determine the nucleotide specificity and dissect the energetics of the binding of MgATP. The order of affinity of various nucleoside triphosphates for HPPK is MgATP>MgGTP>MgITP>MgXTP approximately MgUTP approximately MgCTP. The affinity of MgATP for HPPK (K(d)=2.6+/-0.06 microM) is 260-fold higher than that of MgGTP and more than 1000-fold higher than those of the other nucleoside triphosphates, indicating that HPPK is highly specific with respect to the base moiety of the nucleotide. The affinity of ATP for HPPK in the presence of Mg(2+) is 15 times that in the absence of Mg(2+), indicating that the metal ion is important for the binding of the nucleotide. Removal of the gamma-phosphate from MgATP reduces its affinity for HPPK by a factor of approximately 21. The affinity of AMP for HPPK is about one third that of ADP and almost the same as that of adenosine. The result suggests that among the three phosphoryl groups of MgATP, the gamma-phosphoryl group is most critical for binding to HPPK and the alpha-phosphoryl group contributes little to the binding of the nucleotide. The affinity of MgATP is 18 times that of MgdATP, indicating that the 2'-hydroxyl group of MgATP is also important for binding. van't Hoff analysis suggests that binding of MgATP is mainly driven by enthalpy at 25 degrees C and the entropy of binding is also in favor of the formation of the HPPK.MgATP complex.

The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents.

Background: Folate cofactors are essential for life. Mammals derive folates from their diet, whereas most microorganisms must synthesize folates de novo. Enzymes of the folate pathway therefore provide ideal targets for the development of antimicrobial agents. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate biosynthetic pathway., Results: The crystal structure of HPPK from Escherichia coli has been determined at 1.5 A resolution with a crystallographic R factor of 0.182. The HPPK molecule has a novel three-layered alpha beta alpha fold that creates a valley approximately 26 A long, 10 A wide and 10 A deep. The active center of HPPK is located in the valley and the substrate-binding sites have been identified with the aid of NMR spectroscopy. The HP-binding site is located at one end of the valley, near Asn55, and is sandwiched between two aromatic sidechains. The ATP-binding site is located at the other end of the valley. The adenine base of ATP is positioned near Leu111 and the ribose and the triphosphate extend across and reach the vicinity of HP., Conclusions: The HPPK structure provides a framework to elucidate structure/function relationships of the enzyme and to analyze mechanisms of pyrophosphoryl transfer. Furthermore, this work may prove useful in the structure-based design of new antimicrobial agents.

The folic acid synthesis (Fas) protein of Pneumocystis carinii is a multifunctional enzyme containing dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (PPPK), and dihydropteroate synthase activities. Isolation of the stretch of fas cDNA shown by amino acid similarity to the bacterial counterparts to code for PPPK activity (fasC domain) is described. FasC was expressed to high levels in Escherichia coli inclusion bodies using an inducible tac promoter expression system. Solubilization of the inclusion bodies in 6 M guanidine hydrochloride and refolding of the recombinant protein yielded enzymatically active PPPK which was purified to homogeneity by anion-exchange and gel-filtration chromatography. Sequence analysis showed that the first 13 amino acids of the purified protein were in agreement with those predicted from the DNA sequence and, furthermore, that the amino-terminal methionine had been removed. The enzyme is active in the monomeric form, exhibiting maximum activity at around pH 8.0. Isoelectric focusing gave a pI of 9.1. The Km value for 6-hydroxymethyl-7,8-dihydropterin was 3.6 microM in 50 mM Tris buffer, pH 8.2. The production of independently folded, active P. carinii PPPK will allow detailed biochemical and structural studies, increasing our understanding of this enzyme domain.

The enzyme dihydropteroate synthetase (DHPS) from Plasmodium falciparum is involved in the mechanism of action of the sulfone/sulfonamide group of drugs. We describe the cloning and sequencing of the gene encoding the P. falciparum DHPS enzyme and show that it is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin pyrophosphokinase (PPPK) at the N terminus of DHPS. The gene encodes a putative protein of 83 kDa that contains two domains that are homologous with the DHPS and PPPK enzymes of other organisms. The PPPK-DHPS gene is encoded on chromosome 8 and has two introns. An antibody raised to the PPPK region of the protein was found to recognize a 68-kDa protein that is expressed throughout the asexual life cycle of the parasite. We have determined the sequence of the DHPS portion of the gene from sulfadoxine-sensitive and -resistant P. falciparum clones and identified sequence differences that may have a role in sulfone/sulfonamide resistance.

Streptomyces extracellular nucleotide 3'-pyrophosphokinase-2',3'-cyclic monophosphokinase transfers 5'-beta,gamma-pyrophosphate group from ATP, some ATP derivatives and dATP to a variety of nucleot(s)ides at the 3'-OH site and synthesizes the respective 3'-pyrophosphoryl nucleotides. The enzyme also utilizes A-5'-pn (n = 3-5)-5'-N, transferring their adenosine 5'-pyrophosphoryl group and concomitantly eliminating the AMP moiety therefrom, leading to the synthesis of 2',3'-cyclic monophosphoryl nucleotides. The enzyme gene and its neighbouring up- and downstream sequences were analyzed. Streptomyces and enteric bacteria E. coli and Klebsiella pneumoniae transformants were constructed by incorporation of the pyrophosphokinase gene-recombined expression plasmids. Various effects of the treatments--intracellular synthesis of 3'-pyrophosphoryl nucleotides, retarded cellular growth, stimulated N2 fixation by K. pneumoniae and so on--were seen.