Structural basis for substrate recognition of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis.

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel ß sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.

Closure of the Human TKFC Active Site: Comparison of the Apoenzyme and the Complexes Formed with Either Triokinase or FMN Cyclase Substrates.

Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.

Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase.

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.

Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae.

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported in Saccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid ß-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3-NAD+ complex and the MDH3-NAD+-OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

Transitions in DNA polymerase ß µs-ms dynamics related to substrate binding and catalysis.

DNA polymerase ß (pol ß) plays a central role in the DNA base excision repair pathway and also serves as an important model polymerase. Dynamic characterization of pol ß from methyl-TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of Ile and Met sidechains and previous backbone relaxation dispersion measurements, reveals transitions in µs-ms dynamics in response to highly variable substrates. Recognition of a 1-nt-gapped DNA substrate is accompanied by significant backbone and sidechain motion in the lyase domain and the DNA binding subdomain of the polymerase domain, that may help to facilitate binding of the apoenzyme to the segments of the DNA upstream and downstream from the gap. Backbone µs-ms motion largely disappears after formation of the pol ß-DNA complex, giving rise to an increase in uncoupled µs-ms sidechain motion throughout the enzyme. Formation of an abortive ternary complex using a non-hydrolyzable dNTP results in sidechain motions that fit to a single exchange process localized to the catalytic subdomain, suggesting that this motion may play a role in catalysis.

Isolation and Assays of Bacterial Dimethylsulfoniopropionate Lyases.

The organosulfur metabolite dimethylsulfoniopropionate (DMSP) and its enzymatic breakdown product dimethyl sulfide (DMS) have important implications in the global sulfur cycle and in marine microbial food webs. Enormous amounts of DMSP are produced in marine environments where microbial communities import and catabolize it via either the demethylation or the cleavage pathways. The enzymes that cleave DMSP are termed "DMSP lyases" and generate acrylate or hydroxypropionate, and ~107tons of DMS annually. An important environmental factor affecting DMS generation by the DMSP lyases is the availability of metal ions as these enzymes use various cofactors for catalysis. This chapter summarizes advances on bacterial DMSP catabolism, with an emphasis on various biochemical methods employed for the isolation and characterization of bacterial DMSP lyases. Strategies are presented for the purification of DMSP lyases expressed in bacterial cells. Specific conditions for the efficient isolation of apoproteins in Escherichia coli are detailed. DMSP cleavage is effectively inferred, utilizing the described HPLC-based acrylate detection assay. Finally, substrate and metal binding interactions are examined using fluorescence and UV-visible assays. Together, these methods are rapid and well suited for the biochemical and structural characterization of DMSP lyases and in the assessment of uncharacterized DMSP catabolic enzymes, and new metalloenzymes in general.

Structural and Functional Characterization of Malate Synthase G from Opportunistic Pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg2+ for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg2+. We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.

Activation Mechanism of the Streptomyces Tyrosinase Assisted by the Caddie Protein.

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a "caddie" protein that assists the transport of two copper ions into the active center. In this complex, the Tyr98 residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr98 residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of µ-η2:η2-peroxo-dicopper(II) and Cu(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr98 residue to dopaquinone, may ensure the generation of fully activated tyrosinase.

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/ß-hydrolase superfamily.

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s-1 and 2.5 ± 0.2 × 107 m-1 s-1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/ß-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-ß-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR): tumor expression patterns and prognostic value in oral cancer.

BACKGROUND: Tumor-specific biomarkers are a prerequisite for the development of targeted imaging and therapy in oral squamous cell carcinoma (OSCC). urokinase-type Plasminogen Activator Receptor (uPAR), Tissue Factor (TF) and Epidermal Growth Factor Receptor (EGFR) are three biomarkers that exhibit enhanced expression in many types of cancers, and have been investigated as potential biomarkers for targeted strategies and prognostication. The aim of the study was to investigate the expression patterns of uPAR, TF and EGFR and their potential prognostic value in OSCC. METHODS: Immunohistochemical expression of uPAR, TF and EGFR in tumor resection specimens from 191 patients with primary OSCC was analyzed. Overall (OS) and disease-free survival (DFS) was calculated. Associations between biomarker expression, clinicopathological factors and patient survival was analyzed using the Cox proportional hazards model for univariate and multivariate analysis, log rank and Kaplan-Meier statistics. RESULTS: uPAR and TF exhibited a highly tumor-specific expression pattern while EGFR also showed expression in normal tissues outside the tumor compartment. The overall positive expression rate of uPAR, TF and EGFR was 95%, 58% and 98%, respectively. High uPAR expression across the entire cohort was negatively associated with OS (p = 0.031, HR = 1.595 (95%CI 1.044-2.439)) in univariate analysis. The 5-year OS for high and low uPAR expression was 39% and 56%, respectively. The expression of TF and EGFR was not associated with survival outcome. CONCLUSIONS: This study may suggest that uPAR and TF could potentially be attractive targets for molecular imaging and therapy in OSCC due to high positive expression rates and tumor-specific expression patterns. High uPAR expression was significantly associated with a reduced survival. uPAR seems to be a prognostic biomarker in oral cancer.

Carbohydrate-binding architecture of the multi-modular α-1,6-glucosyltransferase from Paenibacillus sp. 598K, which produces α-1,6-glucosyl-α-glucosaccharides from starch.

Paenibacillus sp. 598K α-1,6-glucosyltransferase (Ps6TG31A), a member of glycoside hydrolase family 31, catalyzes exo-α-glucohydrolysis and transglucosylation and produces α-1,6-glucosyl-α-glucosaccharides from α-glucan via its disproportionation activity. The crystal structure of Ps6TG31A was determined by an anomalous dispersion method using a terbium derivative. The monomeric Ps6TG31A consisted of one catalytic (ß/α)8-barrel domain and six small domains, one on the N-terminal and five on the C-terminal side. The structures of the enzyme complexed with maltohexaose, isomaltohexaose, and acarbose demonstrated that the ligands were observed in the catalytic cleft and the sugar-binding sites of four ß-domains. The catalytic site was structured by a glucose-binding pocket and an aglycon-binding cleft built by two sidewalls. The bound acarbose was located with its non-reducing end pseudosugar docked in the pocket, and the other moieties along one sidewall serving three subsites for the α-1,4-glucan. The bound isomaltooligosaccharide was found on the opposite sidewall, which provided the space for the acceptor molecule to be positioned for attack of the catalytic intermediate covalent complex during transglucosylation. The N-terminal domain recognized the α-1,4-glucan in a surface-binding mode. Two of the five C-terminal domains belong to the carbohydrate-binding modules family 35 and one to family 61. The sugar complex structures indicated that the first family 35 module preferred α-1,6-glucan, whereas the second family 35 module and family 61 module preferred α-1,4-glucan. Ps6TG31A appears to have enhanced transglucosylation activity facilitated by its carbohydrate-binding modules and substrate-binding cleft that positions the substrate and acceptor sugar for the transglucosylation.

ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein.

Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B12, heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism.

The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.

A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding.

The catalytic active site of Mn-specific superoxide dismutase (MnSOD) is organized around a redox-active Mn ion. The most highly conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the active sites of these two types of SODs. Mutation of this Gln69 to Glu in Escherichia coli FeSOD increased the Fe3+/2+ reduction midpoint potential by >0.6 V without disrupting the structure or Fe binding [ Yikilmaz, E., Rodgers, D. W., and Miller, A.-F. ( 2006 ) Biochemistry 45 ( 4 ), 1151 - 1161 ]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °C higher that of wild-type (WT) apoMnSOD, whereas the Tm of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the Tm attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus, our data refute the notion that the WT residues optimize the structural stability of the protein and instead are consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy to favor metal ion binding.

Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation.

Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M ß-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation.

CTX-M ß-lactamases provide resistance against the ß-lactam antibiotic, cefotaxime, but not a related antibiotic, ceftazidime. ß-Lactamases that carry the P167S substitution, however, provide ceftazidime resistance. In this study, CTX-M-14 was used as a model to study the structural changes caused by the P167S mutation that accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the P167S apoenzyme along with the structures of the S70G/P167S, E166A/P167S, and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apoenzyme. The S70G and E166A mutations allow capture of the enzyme-substrate complex and the acylated form of ceftazidime, respectively. The results showed a large conformational change in the Ω-loop of the ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex, suggesting the change occurs upon acylation. The change results in a larger active site that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing accommodation of ceftazidime for hydrolysis. In addition, the conformational change was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure toward a conformational change in the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to an increased level of ceftazidime hydrolysis.

Polycomb repressive complex 2 in an autoinhibited state.

Polycomb-group proteins control many fundamental biological processes, such as anatomical development in mammals and vernalization in plants. Polycomb repressive complex 2 (PRC2) is responsible for methylation of histone H3 lysine 27 (H3K27), and trimethylated H3K27 (H3K27me3) is implicated in epigenetic gene silencing. Recent genomic, biochemical, and structural data indicate that PRC2 is broadly conserved from yeast to human in many aspects. Here, we determined the crystal structure of an apo-PRC2 from the fungus Chaetomium thermophilum captured in a bona fide autoinhibited state, which represents a novel conformation of PRC2 associated with enzyme regulation in light of the basal and stimulated states that we reported previously. We found that binding by the cofactor S-adenosylmethionine mitigates this autoinhibited structural state. Using steady-state enzyme kinetics, we also demonstrated that disrupting the autoinhibition results in a vastly activated enzyme complex. Autoinhibition provides a novel structural platform that may enable control of PRC2 activity in response to diverse transcriptional states and chromatin contexts and set a ground state to allow PRC2 activation by other cellular mechanisms as well.

Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate.

Thymidylate kinase (TMK) is a key enzyme which plays an important role in DNA synthesis. It belongs to the family of nucleoside monophosphate kinases, several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three-dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal-mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild-type protein and mutants. Furthermore, the enzyme was found to exhibit broad specificity toward phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes. DATABASE: Structural data are available in the PDB under the accession numbers 2PBR, 4S2E, 5H5B, 5XAI, 4S35, 5XB2, 5H56, 5XB3, 5H5K, 5XB5, and 5XBH.

Streptococcus pyogenes quinolinate-salvage pathway-structural and functional studies of quinolinate phosphoribosyl transferase and NH3 -dependent NAD+ synthetase.

Streptococcus pyogenes, also known as Group A Strep (GAS), is an obligate human pathogen that is responsible for millions of infections and numerous deaths per year. Infection manifestations can range from simple, acute pharyngitis to more complex, necrotizing fasciitis. To date, most treatments for GAS infections involve the use of common antibiotics including tetracycline and clindamycin. Unfortunately, new strains have been identified that are resistant to these drugs, therefore, new targets must be identified to treat drug-resistant strains. This work is focused on the structural and functional characterization of three proteins: spNadC, spNadD, and spNadE. These enzymes are involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD+ ). The structures of spNadC and spNadE were determined. SpNadC is suggested to play a role in GAS virulence, while spNadE, functions as an NAD synthetase and is considered to be a new drug target. Determination of the spNadE structure uncovered a putative, NH3 channel, which may provide insight into the mechanistic details of NH3 -dependent NAD+ synthetases in prokaryotes. ENZYMES: Quinolinate phosphoribosyltransferase: EC2.4.2.19 and NAD synthetase: EC6.3.1.5. DATABASE: Protein structures for spNadC, spNadCΔ69A , and spNadE are deposited into Protein Data Bank under the accession codes 5HUL, 5HUO & 5HUP, and 5HUH & 5HUJ, respectively.