Self-Immolation of a Bacterial Dehydratase Enzyme by its Epoxide Product.

Disabling the bacterial capacity to cause infection is an innovative approach that has attracted significant attention to fight against superbugs. A relevant target for anti-virulence drug discovery is the type I dehydroquinase (DHQ1) enzyme. It was shown that the 2-hydroxyethylammonium derivative 3 has in vitro activity since it causes the covalent modification of the catalytic lysine residue of DHQ1. As this compound does not bear reactive electrophilic centers, how the chemical modification occurs is intriguing. We report here an integrated approach, which involves biochemical studies, X-ray crystallography and computational studies on the reaction path using combined quantum mechanics/molecular mechanics Umbrella Sampling Molecular Dynamics, that evidences that DHQ1 catalyzes its self-immolation by transforming the unreactive 2-hydroxyethylammonium group in 3 into an epoxide that triggers the lysine covalent modification. This finding might open opportunities for the design of lysine-targeted irreversible inhibitors bearing a 2-hydroxyethylammonium moiety as an epoxide proform, which to our knowledge has not been reported previously.

Crystal structure of NucB, a biofilm-degrading endonuclease.

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ßßα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

Freezing the Dynamic Gap for Selectivity: Motion-Based Design of Inhibitors of the Shikimate Kinase Enzyme.

Shikimate kinase (SK), the fifth enzyme of the aromatic amino acid biosynthesis, is a recognized target for antibiotic drug discovery. The potential of the distinct dynamic apolar gap, which isolates the natural substrate from the solvent environment for catalysis, and the motion of Mycobacterium tuberculosis and Helicobacter pylori SK enzymes, which was observed by molecular dynamics simulations, was explored for inhibition selectivity. The results of the biochemical and computational studies reveal that the incorporation of bulky groups at position C5 of 5-aminoshikimic acid and the natural substrate enhances the selectivity for the H. pylori enzyme due to key motion differences in the shikimic acid binding domain (mainly helix α5). These studies show that the less-exploited motion-based design approach not only is an alternative strategy for the development of competitive inhibitors, but could also be a way to achieve selectivity against a particular enzyme among its homologues.

Study of the Phosphoryl-Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy.

The phosphoryl-transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D (1)H and (31)P NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ-phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure-based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymes.

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct.

The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzyme.

Irreversible covalent modification of type I dehydroquinase with a stable Schiff base.

The irreversible inhibition of type I dehydroquinase (DHQ1), the third enzyme of the shikimic acid pathway, is investigated by structural, biochemical and computational studies. Two epoxides, which are mimetics of the natural substrate, were designed as irreversible inhibitors of the DHQ1 enzyme and to study the binding requirements of the linkage to the enzyme. The epoxide with the S configuration caused the covalent modification of the protein whereas no reaction was obtained with its epimer. The first crystal structure of DHQ1 from Salmonella typhi covalently modified by the S epoxide, which is reported at 1.4 Å, revealed that the modified ligand is surprisingly covalently attached to the essential Lys170 by the formation of a stable Schiff base. The experimental and molecular dynamics simulation studies reported here highlight the huge importance of the conformation of the C3 carbon of the ligand for covalent linkage to this type of aldolase I enzyme, revealed the key role played by the essential His143 as a Lewis acid in this process and show the need for a neatly closed active site for catalysis.

Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases.

DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymes.

Insights into substrate binding and catalysis in bacterial type I dehydroquinase.

Structural, biochemical and computational studies to study substrate binding and the role of the conserved residues of the DHQ1 (type I dehydroquinase) enzyme active site are reported in the present paper. The crystal structure of DHQ1 from Salmonella typhi in complex with (2R)-2-methyl-3-dehydroquinic acid, a substrate analogue, was solved at 1.5 Å. The present study reveals a previously unknown key role for conserved Glu46, Phe145 and Met205 and Gln236, Pro234 and Ala233 residues, with the latter three being located in the flexible substrate-covering loop. Gln236 was shown to be responsible for the folding of this loop and for the dramatic reduction of its flexibility, which triggers active site closure. Glu46 was found to be key in bringing the substrate close to the lysine/histidine catalytic pocket to initiate catalysis. The present study could be useful in the rational design of inhibitors of this challenging and recognized target for the development of novel herbicides and antimicrobial agents.

Mycobacterium tuberculosis shikimate kinase inhibitors: design and simulation studies of the catalytic turnover.

Shikimate kinase (SK) is an essential enzyme in several pathogenic bacteria and does not have any counterpart in human cells, thus making it an attractive target for the development of new antibiotics. The key interactions of the substrate and product binding and the enzyme movements that are essential for catalytic turnover of the Mycobacterium tuberculosis shikimate kinase enzyme (Mt-SK) have been investigated by structural and computational studies. Based on these studies several substrate analogs were designed and assayed. The crystal structure of Mt-SK in complex with ADP and one of the most potent inhibitors has been solved at 2.15 Å. These studies reveal that the fixation of the diaxial conformation of the C4 and C5 hydroxyl groups recognized by the enzyme or the replacement of the C3 hydroxyl group in the natural substrate by an amino group is a promising strategy for inhibition because it causes a dramatic reduction of the flexibility of the LID and shikimic acid binding domains. Molecular dynamics simulation studies showed that the product is expelled from the active site by three arginines (Arg117, Arg136, and Arg58). This finding represents a previously unknown key role of these conserved residues. These studies highlight the key role of the shikimic acid binding domain in the catalysis and provide guidance for future inhibitor designs.

Quinate-based ligands for irreversible inactivation of the bacterial virulence factor DHQ1 enzyme-A molecular insight.

Irreversible inhibition of the enzyme type I dehydroquinase (DHQ1), a promising target for anti-virulence drug development, has been explored by enhancing the electrophilicity of specific positions of the ligand towards covalent lysine modification. For ligand design, we made use of the advantages offered by the intrinsic acid-base properties of the amino substituents introduced in the quinate scaffold, namely compounds 6-7 (R configuration at C3), to generate a potential leaving group, as well as the recognition pattern of the enzyme. The reactivity of the C2-C3 bond (Re face) in the scaffold was also explored using compound 8. The results of the present study show that replacement of the C3 hydroxy group of (-)-quinic acid by a hydroxyamino substituent (compound 6) provides a time-dependent irreversible inhibitor, while compound 7, in which the latter functionality was substituted by an amino group, and the introduction of an oxirane ring at C2-C3 bond, compound 8, do not allow covalent modification of the enzyme. These outcomes were supported by resolution of the crystal structures of DHQ1 from Staphylococcus aureus (Sa-DHQ1) and Salmonella typhi (St-DHQ1) chemically modified by 6 at a resolution of 1.65 and 1.90 Å, respectively, and of St-DHQ1 in the complex with 8 (1.55 Å). The combination of these structural studies with extensive molecular dynamics simulation studies allowed us to understand the molecular basis of the type of inhibition observed. This study is a good example of the importance of achieving the correct geometry between the reactive center of the ligand (electrophile) and the enzyme nucleophile (lysine residue) to allow selective covalent modification. The outcomes obtained with the hydroxyamino derivative 6 also open up new possibilities in the design of irreversible inhibitors based on the use of amino substituents.

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency.

Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.

Functional analysis of the GTPases EngA and YhbZ encoded by Salmonella typhimurium.

The S. typhimurium genome encodes proteins, designated EngA and YhbZ, which have a high sequence identity with the GTPases EngA/Der and ObgE/CgtAE of Escherichia coli. The wild-type activity of the E. coli proteins is essential for normal ribosome maturation and cell viability. In order to characterize the potential involvement of the Salmonella typhimurium EngA and YhbZ proteins in ribosome biology, we used high stringency affinity chromatography experiments to identify strongly binding ribosomal partner proteins. A combination of biochemical and microcalorimetric analysis was then used to characterize these protein:protein interactions and quantify nucleotide binding affinities. These experiments show that YhbZ specifically interacts with the pseudouridine synthase RluD (KD=2 microM and 1:1 stoichiometry), and we show for the first time that EngA can interact with the ribosomal structural protein S7. Thermodynamic analysis shows both EngA and YhbZ bind GDP with a higher affinity than GTP (20-fold difference for EngA and 3.8-fold for YhbZ), and that the two nucleotide binding sites in EngA show a 5.3-fold difference in affinity for GDP. We report a fluorescence assay for nucleotide binding to EngA and YhbZ, which is suitable for identifying inhibitors specific for this ligand-binding site, which would potentially inhibit their biological functions. The interactions of YhbZ with ribosome structural proteins that we identify may demonstrate a previously unreported additional function for this class of GTPase: that of ensuring delivery of rRNA modifying enzymes to the appropriate region of the ribosome.

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases.

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.

Reducing the Flexibility of Type†II Dehydroquinase for Inhibition: A Fragment-Based Approach and Molecular Dynamics Study.

A multidisciplinary approach was used to identify and optimize a quinazolinedione-based ligand that would decrease the flexibility of the substrate-covering loop (catalytic loop) of the type II dehydroquinase from Helicobacter pylori. This enzyme, which is essential for the survival of this bacterium, is involved in the biosynthesis of aromatic amino acids. A computer-aided fragment-based protocol (ALTA) was first used to identify the aromatic fragments able to block the interface pocket that separates two neighboring enzyme subunits and is located at the active site entrance. Chemical modification of its non-aromatic moiety through an olefin cross-metathesis and Seebach's self-reproduction of chirality synthetic principle allowed the development of a quinazolinedione derivative that disables the catalytic loop plasticity, which is essential for the enzyme's catalytic cycle. Molecular dynamics simulations revealed that the ligand would force the catalytic loop into an inappropriate arrangement for catalysis by strong interactions with the catalytic tyrosine and by expelling the essential arginine out of the active site.

Matriptase Induction of Metalloproteinase-Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms.

OBJECTIVE: To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. METHODS: Aggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. RESULTS: The addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage. CONCLUSION: Matriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix alpha3 shifting position initially, followed by movement of alpha2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis-proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.

Structure of Staphylococcus aureus cytidine monophosphate kinase in complex with cytidine 5'-monophosphate.

The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 5'-monophosphate (CMP) has been determined at 2.3 angstroms resolution. The active site reveals novel features when compared with two orthologues of known structure. Compared with the Streptococcus pneumoniae CMK solution structure of the enzyme alone, S. aureus CMK adopts a more closed conformation, with the NMP-binding domain rotating by approximately 16 degrees towards the central pocket of the molecule, thereby assembling the active site. Comparing Escherichia coli and S. aureus CMK-CMP complex structures reveals differences within the active site, including a previously unreported indirect interaction of CMP with Asp33, the replacement of a serine residue involved in the binding of CDP by Ala12 in S. aureus CMK and an additional sulfate ion in the E. coli CMK active site. The detailed understanding of the stereochemistry of CMP binding to CMK will assist in the design of novel inhibitors of the enzyme. Inhibitors are required to treat the widespread hospital infection methicillin-resistant S. aureus (MRSA), currently a major public health concern.

Structure of Staphylococcus aureus guanylate monophosphate kinase.

Nucleotide monophosphate kinases (NMPKs) are potential antimicrobial drug targets owing to their role in supplying DNA and RNA precursors. The present work reports the crystal structure of Staphylococcus aureus guanylate monophosphate kinase (SaGMK) at 1.9 A resolution. The structure shows that unlike most GMKs SaGMK is dimeric, confirming the role of the extended C-terminus in dimer formation as first observed for Escherichia coli GMK (EcGMK). One of the two SaGMK dimers within the crystal asymmetric unit has two monomers in different conformations: an open form with a bound sulfate ion (mimicking the beta-phosphate of ATP) and a closed form with bound GMP and sulfate ion. GMP-induced domain movements in SaGMK can thus be defined by comparison of these conformational states. Like other GMKs, the binding of GMP firstly triggers a partial closure of the enzyme, diminishing the distance between the GMP-binding and ATP-binding sites. In addition, the closed structure shows the presence of a potassium ion in contact with the guanine ring of GMP. The potassium ion appears to form an integral part of the GMP-binding site, as the Tyr36 side chain has significantly moved to form a metal ion-ligand coordination involving the lone pair of the side-chain O atom. The potassium-binding site might also be exploited in the design of novel inhibitors.

Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus.

Prokaryotic genes related to the oxygenase domain of mammalian nitric oxide synthases (NOSs) have recently been identified. Although they catalyze the same reaction as the eukaryotic NOS oxygenase domain, their biological function(s) are unknown. In order to explore rationally the biochemistry and evolution of the prokaryotic NOS family, we have determined the crystal structure of SANOS, from methicillin-resistant Staphylococcus aureus (MRSA), to 2.4 A. Haem and S-ethylisothiourea (SEITU) are bound at the SANOS active site, while the intersubunit site, occupied by the redox cofactor tetrahydrobiopterin (H(4)B) in mammalian NOSs, has NAD(+) bound in SANOS. In common with all bacterial NOSs, SANOS lacks the N-terminal extension responsible for stable dimerization in mammalian isoforms, but has alternative interactions to promote dimer formation.