Crystal Structure, Steady-State, and Pre-Steady-State Kinetics of Acinetobacter baumannii ATP Phosphoribosyltransferase.

The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N1-(5-phospho-ß-d-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisGS, is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in kcat. Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisGS) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, kcat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisGS. The HisGS-catalyzed reaction is limited by the chemical step, as suggested by the enhancement of kcat when Mg2+ was replaced by Mn2+, and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than kcat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.

Non-canonical binding of the Chaetomium thermophilum PolD4 N-terminal PIP motif to PCNA involves Q-pocket and compact 2-fork plug interactions but no 310 helix.

DNA polymerase δ (Pol δ) is a key enzyme for the maintenance of genome integrity in eukaryotic cells, acting in concert with the sliding clamp processivity factor PCNA (proliferating cell nuclear antigen). Three of the four subunits of human Pol δ interact directly with the PCNA homotrimer via a short, conserved protein sequence known as a PCNA interacting protein (PIP) motif. Here, we describe the identification of a PIP motif located towards the N terminus of the PolD4 subunit of Pol δ (equivalent to human p12) from the thermophilic filamentous fungus Chaetomium thermophilum and present the X-ray crystal structure of the corresponding peptide bound to PCNA at 2.45 Å. Like human p12, the fungal PolD4 PIP motif displays non-canonical binding to PCNA. However, the structures of the human p12 and fungal PolD4 PIP motif peptides are quite distinct, with the fungal PolD4 PIP motif lacking the 310 helical segment that characterises most previously identified PIP motifs. Instead, the fungal PolD4 PIP motif binds PCNA via conserved glutamine that inserts into the Q-pocket on the surface of PCNA and with conserved leucine and phenylalanine sidechains forming a compact 2-fork plug that inserts into the hydrophobic pocket on PCNA. Despite the unusual binding mode of the fungal PolD4, isothermal calorimetry (ITC) measurements show that its affinity for PCNA is similar to that of its human orthologue. These observations add to a growing body of information on how diverse proteins interact with PCNA and highlight how binding modes can vary significantly between orthologous PCNA partner proteins.

Canonical binding of Chaetomium thermophilum DNA polymerase δ/ζ subunit PolD3 and flap endonuclease Fen1 to PCNA.

The sliding clamp PCNA is a key player in eukaryotic genome replication and stability, acting as a platform onto which components of the DNA replication and repair machinery are assembled. Interactions with PCNA are frequently mediated via a short protein sequence motif known as the PCNA-interacting protein (PIP) motif. Here we describe the binding mode of a PIP motif peptide derived from C-terminus of the PolD3 protein from the thermophilic ascomycete fungus C. thermophilum, a subunit of both DNA polymerase δ (Pol δ) and the translesion DNA synthesis polymerase Pol ζ, characterised by isothermal titration calorimetry (ITC) and protein X-ray crystallography. In sharp contrast to the previously determined structure of a Chaetomium thermophilum PolD4 peptide bound to PCNA, binding of the PolD3 peptide is strictly canonical, with the peptide adopting the anticipated 310 helix structure, conserved Gln441 inserting into the so-called Q-pocket on PCNA, and Ile444 and Phe448 forming a two-fork plug that inserts into the hydrophobic surface pocket on PCNA. The binding affinity for the canonical PolD3 PIP-PCNA interaction determined by ITC is broadly similar to that previously determined for the non-canonical PolD4 PIP-PCNA interaction. In addition, we report the structure of a PIP peptide derived from the C. thermophilum Fen1 nuclease bound to PCNA. Like PolD3, Fen1 PIP peptide binding to PCNA is achieved by strictly canonical means. Taken together, these results add to an increasing body of information on how different proteins bind to PCNA, both within and across species.

Allosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation.

ATP phosphoribosyltransferase catalyses the first step of histidine biosynthesis and is controlled via a complex allosteric mechanism where the regulatory protein HisZ enhances catalysis by the catalytic protein HisGS while mediating allosteric inhibition by histidine. Activation by HisZ was proposed to position HisGS Arg56 to stabilise departure of the pyrophosphate leaving group. Here we report active-site mutants of HisGS with impaired reaction chemistry which can be allosterically restored by HisZ despite the HisZ:HisGS interface lying ~20 Å away from the active site. MD simulations indicate HisZ binding constrains the dynamics of HisGS to favour a preorganised active site where both Arg56 and Arg32 are poised to stabilise leaving-group departure in WT-HisGS. In the Arg56Ala-HisGS mutant, HisZ modulates Arg32 dynamics so that it can partially compensate for the absence of Arg56. These results illustrate how remote protein-protein interactions translate into catalytic resilience by restoring damaged electrostatic preorganisation at the active site.

Next generation Glucose-1-phosphate thymidylyltransferase (RmlA) inhibitors: An extended SAR study to direct future design.

The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor's pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.

Mapping the Structural Path for Allosteric Inhibition of a Short-Form ATP Phosphoribosyltransferase by Histidine.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis, being allosterically inhibited by the final product of the pathway. Allosteric inhibition of long-form ATPPRTs by histidine has been extensively studied, but inhibition of short-form ATPPRTs is poorly understood. Short-form ATPPRTs are hetero-octamers formed by four catalytic subunits (HisGS) and four regulatory subunits (HisZ). HisGS alone is catalytically active and insensitive to histidine. HisZ enhances catalysis by HisGS in the absence of histidine but mediates allosteric inhibition in its presence. Here, steady-state and pre-steady-state kinetics establish that histidine is a noncompetitive inhibitor of short-form ATPPRT and that inhibition does not occur by dissociating HisGS from the hetero-octamer. The crystal structure of ATPPRT in complex with histidine and the substrate 5-phospho-α-d-ribosyl-1-pyrophosphate was determined, showing histidine bound solely to HisZ, with four histidine molecules per hetero-octamer. Histidine binding involves the repositioning of two HisZ loops. The histidine-binding loop moves closer to histidine to establish polar contacts. This leads to a hydrogen bond between its Tyr263 and His104 in the Asp101-Leu117 loop. The Asp101-Leu117 loop leads to the HisZ-HisGS interface, and in the absence of histidine, its motion prompts HisGS conformational changes responsible for catalytic activation. Following histidine binding, interaction with the histidine-binding loop may prevent the Asp101-Leu117 loop from efficiently sampling conformations conducive to catalytic activation. Tyr263Phe-PaHisZ-containing PaATPPRT, however, is less susceptible though not insensitive to histidine inhibition, suggesting the Tyr263-His104 interaction may be relevant to yet not solely responsible for transmission of the allosteric signal.

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-ß-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.

Structural insights into the mechanism and inhibition of the ß-hydroxydecanoyl-acyl carrier protein dehydratase from Pseudomonas aeruginosa.

Fatty acid biosynthesis is an essential component of metabolism in both eukaryotes and prokaryotes. The fatty acid biosynthetic pathway of Gram-negative bacteria is an established therapeutic target. Two homologous enzymes FabA and FabZ catalyze a key step in fatty acid biosynthesis; both dehydrate hydroxyacyl fatty acids that are coupled via a phosphopantetheine to an acyl carrier protein (ACP). The resulting trans-2-enoyl-ACP is further polymerized in a processive manner. FabA, however, carries out a second reaction involving isomerization of trans-2-enoyl fatty acid to cis-3-enoyl fatty acid. We have solved the structure of Pseudomonas aeruginosa FabA with a substrate allowing detailed molecular insight into the interactions of the active site. This has allowed a detailed examination of the factors governing the second catalytic step. We have also determined the structure of FabA in complex with small molecules (so-called fragments). These small molecules occupy distinct regions of the active site and form the basis for a rational inhibitor design program.

Allosteric competitive inhibitors of the glucose-1-phosphate thymidylyltransferase (RmlA) from Pseudomonas aeruginosa.

Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.

Chemical, genetic and structural assessment of pyridoxal kinase as a drug target in the African trypanosome.

Pyridoxal-5'-phosphate (vitamin B(6) ) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B(6) de novo and rely on uptake of B(6) vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg(-1) and a K(m)(app) with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease.

Dihydroquinazolines as a novel class of Trypanosoma brucei trypanothione reductase inhibitors: discovery, synthesis, and characterization of their binding mode by protein crystallography.

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei , the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR-ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

Structure of Trypanosoma brucei glutathione synthetase: domain and loop alterations in the catalytic cycle of a highly conserved enzyme.

Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-gamma-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery.

Inhibitors of the kinase IspE: structure-activity relationships and co-crystal structure analysis.

Enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are therapeutic targets for the treatment of important infectious diseases. Whereas this pathway is absent in humans, it is used by plants, many eubacteria and apicomplexan protozoa, including major human pathogens such as Plasmodium falciparum and Mycobacterium tuberculosis. Herein, we report on the design, preparation and biological evaluation of a new series of ligands for IspE protein, a kinase from this pathway. These inhibitors were developed for the inhibition of IspE from Escherichia coli, using structure-based design approaches. Structure-activity relationships (SARs) and a co-crystal structure of Aquifex aeolicus IspE bound to a representative inhibitor validate the proposed binding mode. The crystal structure shows that the ligand binds in the substrate-rather than the adenosine 5'-triphosphate (ATP)-binding pocket. As predicted, a cyclopropyl substituent occupies a small cavity not used by the substrate. The optimal volume occupancy of this cavity is explored in detail. In the co-crystal structure, a diphosphate anion binds to the Gly-rich loop, which normally accepts the triphosphate moiety of ATP. This structure provides useful insights for future structure-based developments of inhibitors for the parasite enzymes.

Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases.

TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite K(m) and V(max) values. The structure of the reduced enzyme at 2.1 A (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 A (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.

The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation.

A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase - ligand recognition in a template for antimicrobial drug discovery.

4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP alpha-phosphate not the binding site for the methyl-D-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.

Synthesis and characterization of cytidine derivatives that inhibit the kinase IspE of the non-mevalonate pathway for isoprenoid biosynthesis.

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co-crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co-crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well-conserved ATP-binding Gly-rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure-based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.

Crystal structures of Trypanosoma brucei and Staphylococcus aureus mevalonate diphosphate decarboxylase inform on the determinants of specificity and reactivity.

Mevalonate diphosphate decarboxylase (MDD) catalyzes the ATP-dependent decarboxylation of mevalonate 5-diphosphate (MDP) to form isopentenyl pyrophosphate, a ubiquitous precursor for isoprenoid biosynthesis. MDD is a poorly understood component of this important metabolic pathway. Complementation of a temperature-sensitive yeast mutant by the putative mdd genes of Trypanosoma brucei and Staphylococcus aureus provides proof-of-function. Crystal structures of MDD from T. brucei (TbMDD, at 1.8 A resolution) and S. aureus (SaMDD, in two distinct crystal forms, each diffracting to 2.3 A resolution) have been determined. Gel-filtration chromatography and analytical ultracentrifugation experiments indicate that TbMDD is predominantly monomeric in solution while SaMDD is dimeric. The new crystal structures and comparison with that of the yeast Saccharomyces cerevisiae enzyme (ScMDD) reveal the structural basis for this variance in quaternary structure. The presence of an ordered sulfate in the structure of TbMDD reveals for the first time details of a ligand binding in the MDD active site and, in conjunction with well-ordered water molecules, comparisons with the related enzyme mevalonate kinase, structural and biochemical data derived on ScMDD and SaMDD, allows us to model a ternary complex with MDP and ATP. This model facilitates discussion of the molecular determinants of substrate recognition and contributions made by specific residues to the enzyme mechanism.

Trypanosoma brucei UDP-galactose-4'-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-alpha-D-galactose.

The structure of the NAD-dependent oxidoreductase UDP-galactose-4'-epimerase from Trypanosoma brucei in complex with cofactor and the substrate analogue UDP-4-deoxy-4-fluoro-alpha-D-galactose has been determined using diffraction data to 2.7 A resolution. Despite the high level of sequence and structure conservation between the trypanosomatid enzyme and those from humans, yeast and bacteria, the binding of the 4-fluoro-alpha-D-galactose moiety is distinct from previously reported structures. Of particular note is the observation that when bound to the T. brucei enzyme, the galactose moiety of this fluoro-derivative is rotated approximately 180 degrees with respect to the orientation of the hexose component of UDP-glucose when in complex with the human enzyme. The architecture of the catalytic centre is designed to effectively bind different orientations of the hexose, a finding that is consistent with a mechanism that requires the sugar to maintain a degree of flexibility within the active site.

High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei.

Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 A, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Comparisons reveal that these peptides bind in the active site in a manner similar to that of the human cysteine protease inhibitor stefin B when it is complexed to papain. The assignment of the fragment sequences is consistent with the specificity of the protease.