Thesis (Ph. D.)--University of Canterbury, 2007. / Typescript (photocopy). Includes bibliographical references. Also available via the World Wide Web.

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium. Initial investigation into this enzyme categorized it as a methyltransferase. Further investigation revealed that this was incorrect, and that MosA is actually a dihydrodipicolinate synthase, part of the N-acetylneuraminate lyase superfamily. One of the characteristics of enzyme superfamilies is their low sequence identity, but relatively high structural similarity. The structural investigation reported here confirms the high structural similarity between MosA and other superfamily members. <p>Investigation of MosA was carried out by means of x-ray crystallography. It was believed that detailed structural information may shed light into not only the enzymatic mechanism, but also the inhibition of MosA by lysine, the final product of the enzymatic pathway. Insight into enzyme mechanism and inhibition may ultimately prove useful in herbicide or insecticide development, as other dihydrodipicolinate synthases from harmful fungi, bacteria, or plants, make attractive targets for inhibition. Lysine is an essential amino acid for humans, meaning that there is no endogenous lysine production to block the use of these hypothetical inhibitors. Specific inhibitors based on crystal structures have proven to be effective in the past and hopefully, will continue to be useful in the future. <p>Here we report the structure of MosA, solved to 1.95 Å resolution with lysine 161 forming a Schiff-base adduct with pyruvate. This adduct is consistent with the currently accepted dihydrodipicolinate synthase enzyme mechanism.

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium. Initial investigation into this enzyme categorized it as a methyltransferase. Further investigation revealed that this was incorrect, and that MosA is actually a dihydrodipicolinate synthase, part of the N-acetylneuraminate lyase superfamily. One of the characteristics of enzyme superfamilies is their low sequence identity, but relatively high structural similarity. The structural investigation reported here confirms the high structural similarity between MosA and other superfamily members. <p>Investigation of MosA was carried out by means of x-ray crystallography. It was believed that detailed structural information may shed light into not only the enzymatic mechanism, but also the inhibition of MosA by lysine, the final product of the enzymatic pathway. Insight into enzyme mechanism and inhibition may ultimately prove useful in herbicide or insecticide development, as other dihydrodipicolinate synthases from harmful fungi, bacteria, or plants, make attractive targets for inhibition. Lysine is an essential amino acid for humans, meaning that there is no endogenous lysine production to block the use of these hypothetical inhibitors. Specific inhibitors based on crystal structures have proven to be effective in the past and hopefully, will continue to be useful in the future. <p>Here we report the structure of MosA, solved to 1.95 Å resolution with lysine 161 forming a Schiff-base adduct with pyruvate. This adduct is consistent with the currently accepted dihydrodipicolinate synthase enzyme mechanism.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of L-lysine and meso-diaminopimelate biosynthesis, which is the condensation of (S)-aspartate-β-semialdehyde (ASA) and pyruvate into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS has been an attractive antibiotic target because L-lysine and meso-diaminopimelate are cross-linking components between peptidoglycan heteropolysaccharide chains in bacterial cell walls. Studies revealed that mutant auxotrophs for diaminopimelate undergo lysis in the absence of diaminopimelate in the medium; therefore the assumption is that strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and L-lysine biosynthesis in bacteria and would stop or decrease bacterial growth (eventually leading to bacterial death). In this work, the DHDPS inhibitor design is focused on the allosteric site of the enzyme. It was proposed that a compound mimicking binding of two L-lysine molecules at the allosteric site at the enzyme’s dimer-dimer interface would be a more potent inhibitor than the natural allosteric inhibitor of this enzyme, L-lysine. This inhibitor (R,R-bislysine) was synthesized as a racemic mixture, which was then separated with the aid of chiral HPLC. The mechanism of feedback inhibition of DHDPS from Campylobacter jejuni with its natural allosteric modulator, L-lysine, and its synthetic mimic, R,R-bislysine, is studied in detail. It is found that L-lysine is a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. R,R-bislysine is a mixed partial inhibitor with respect to pyruvate and a noncompetitive partial inhibitor with respect to ASA, with an inhibition constant of 200 nM. Kinetic evaluation of each DHDPS mutants (Y110F, H56A, H56N, H59A and H59N) has revealed amino acids responsible for the inhibitory effect of L-lysine, R,R-bislysine, and we have found that R,R-bislysine is a strong submicromolar inhibitor of Y110F, H56A, H56N and H59N.

Dihydrodipicolinate Synthase (EC 4.3.3.7; DHDPS), the product of the dapA gene, is an enzyme that catalyzes the condensation of pyruvate and S-aspartate-β-semialdehyde (ASA) into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS catalyzes the first committed step in the biosynthesis of ʟ-lysine and meso-diaminopimelate; each of which is a necessary cross-linking component between peptidoglycan heteropolysacharide chains of bacterial cell walls. Therefore, strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and ʟ-lysine biosynthesis in bacteria leading to decreased bacterial growth and cell lysis. Much attention has been given to targeting the active site for inhibition; however DHDPS is subject to natural feedback inhibition by ʟ-lysine at an allosteric site. In DHDPS from Campylobacter jejuni ʟ-lysine is known to act as a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. Little is known about how the protein structure facilitates the natural inhibition mechanism and mode of allosteric signal transduction. This work presents ten high resolution crystal structures of Cj-DHDPS and the mutant Y110F-DHDPS with various substrates and inhibitors, including the first reported structure of DHDPS with ASA bound to the active site. As a body of work these structures reveal residues and conformational changes which contribute to the inhibition of the enzyme. Understanding these structure function relationships will be valuable for the design of future antibiotic lead compounds. When an inhibitor binds to the allosteric site there is meaningful shrinkage in the solvent accessible volume between 33% and 49% proportional to the strength of inhibition. Meanwhile at the active site the solvent accessible volume increases between 5% and 35% proportional to the strength of inhibition. Furthermore, inhibitor binding at the allosteric site consistently alters the distance between hydroxyls of the catalytic triad (Y137-T47-Y111') which is likely to affect local pKa's. Changes in active site volume and modification of the catalytic triad would inhibit the enzyme during the binding and condensation of ASA. The residues H56, E88, R60 form a network of hydrogen bonds to close the allosteric site around the inhibitor and act as a lid. Comparison of ʟ-lysine and bislysine bound to wt-DHDPS and Y110F-DHDPS indicates that enhanced inhibition of bislysine is most likely due to increased binding strength rather than altering the mechanism of inhibition. When ASA binds to the active site the network of hydrogen bonds among H56, E88 and R60 is disrupted and the solvent accessible volume of the allosteric site expands by 46%. This observation provides some explanation for the reduced affinity of ʟ-lysine in high ASA concentrations. ʟ-Lysine, but not other inhibitors, is found to induce dynamic domain movements in the wt-DHDPS. These domain movements do not appear to be essential to the inhibition of the enzyme but may play a role in cooperativity between monomers or governing protein dynamics. The moving domain connects the allosteric site to the dimer-dimer interface. Several residues at the weak dimer interface have been identified as potentially involved in dimer-dimer communication including: I172, D173, V176, I194, Y196, S200, N201, K234, D238, Y241, N242 and K245. These residues are not among any previously identified as important for formation of the quaternary structure.

MosA is a protein found in <i>Sinorhizobium meliloti</i> L5-30 and has been suggested to be responsible for the biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine (3-MSI) from scyllo-inosamine (SI). However, we have shown MosA is a dihydrodipicolinate synthase (DHDPS) catalyzing the condensation of pyruvate with aspartate-β-semialdehyde (ASA). Since the DHDPS reaction occurs through a Schiff base aldol-type mechanism it was proposed that MosA could be an O-methyltransferase utilizing 2-oxo-butyrate (2-OB) as a novel methyl donor. This interesting yet unlikely possibility would explain MosA's role in the biosynthesis of 3-MSI without ignoring its similarity to DHDPS. Alternatively, MosA may have two catalytic domains one of which possesses a novel binding motif for S-Adenosyl methionine (SAM) to account for methyltransfer activity. In vitro demonstration of MosAs methyltransferase activity is required to resolve this apparent contradiction.<p>This dissertation describes the chemical synthesis of the rhizopines, investigation into whether MosA has a direct role in rhizopine biosynthesis and the thermodynamic characterization of compounds interacting with MosA as observed by isothermal titration calorimetry. <p>Initial investigation into MosAs methyltransferase activity began with 2-OBs interaction with the enzyme. Inhibition experiments determined 2-OB is a competitive inhibitor with respect to pyruvate of the DHDPS reaction of MosA. Furthermore, protein mass spectrometry of MosA in the presence of 2-OB and sodium borohydride indicated that a Schiff base enzyme intermediate was indeed being formed providing evidence that the proposed mechanism may exist. However, neither of the rhizopines had any effect on the DHDPS activity and HPLC assays determined that no 3-MSI was being produced by MosA in the presence of SI and 2-OB. Furthermore, HPLC assays failed to detect methyl transfer activity by MosA utilizing the SAM as a methyl donor. <p>Isothermal titration calorimetry provided thermodynamic characterization of the pyruvate and 2-OB Schiff base intermediates formed with MosA. In addition, ITC provided insight into the nature and thermodynamics of (S)-lysines inhibition of MosA. ITC failed to detect any interactions between the rhizopines or SAM with MosA. These results indicate that MosA is only a DHDPS and does not catalyze the formation of 3-MSI from SI as hypothesized in the literature.

MosA is a protein found in <i>Sinorhizobium meliloti</i> L5-30 and has been suggested to be responsible for the biosynthesis of the rhizopine 3-O-methyl-scyllo-inosamine (3-MSI) from scyllo-inosamine (SI). However, we have shown MosA is a dihydrodipicolinate synthase (DHDPS) catalyzing the condensation of pyruvate with aspartate-β-semialdehyde (ASA). Since the DHDPS reaction occurs through a Schiff base aldol-type mechanism it was proposed that MosA could be an O-methyltransferase utilizing 2-oxo-butyrate (2-OB) as a novel methyl donor. This interesting yet unlikely possibility would explain MosA's role in the biosynthesis of 3-MSI without ignoring its similarity to DHDPS. Alternatively, MosA may have two catalytic domains one of which possesses a novel binding motif for S-Adenosyl methionine (SAM) to account for methyltransfer activity. In vitro demonstration of MosAs methyltransferase activity is required to resolve this apparent contradiction.<p>This dissertation describes the chemical synthesis of the rhizopines, investigation into whether MosA has a direct role in rhizopine biosynthesis and the thermodynamic characterization of compounds interacting with MosA as observed by isothermal titration calorimetry. <p>Initial investigation into MosAs methyltransferase activity began with 2-OBs interaction with the enzyme. Inhibition experiments determined 2-OB is a competitive inhibitor with respect to pyruvate of the DHDPS reaction of MosA. Furthermore, protein mass spectrometry of MosA in the presence of 2-OB and sodium borohydride indicated that a Schiff base enzyme intermediate was indeed being formed providing evidence that the proposed mechanism may exist. However, neither of the rhizopines had any effect on the DHDPS activity and HPLC assays determined that no 3-MSI was being produced by MosA in the presence of SI and 2-OB. Furthermore, HPLC assays failed to detect methyl transfer activity by MosA utilizing the SAM as a methyl donor. <p>Isothermal titration calorimetry provided thermodynamic characterization of the pyruvate and 2-OB Schiff base intermediates formed with MosA. In addition, ITC provided insight into the nature and thermodynamics of (S)-lysines inhibition of MosA. ITC failed to detect any interactions between the rhizopines or SAM with MosA. These results indicate that MosA is only a DHDPS and does not catalyze the formation of 3-MSI from SI as hypothesized in the literature.

This thesis examined the enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from the pathogen Mycobacterium tuberculosis. DHDPS is a validated antibiotic target for which no potent inhibitor based on substrates, intermediates or product has been found. The importance of the homotetrameric quaternary structure in E. coli DHDPS has been demonstrated by the 100-fold decrease in activity observed in a dimeric variant, DHDPS-L197Y, created by site-directed mutagenesis. This suggested a new approach for inhibitor design: targeting the dimer-dimer interface and disrupting tetramer formation. DHDPS catalyzes the first committed step in the biosynthetic pathway of meso-diaminopimelic acid, a critical component of the mycobacterial cell wall. In this study, wild-type M. tuberculosis DHDPS was thoroughly characterized and compared with the E. coli enzyme. A coupled assay was used to obtain the kinetic parameters for M. tuberculosis DHDPS: KM(S) ASA = 0.43 (±0.02) mM, KMpyruvate = 0.17 (±0.01) mM, and kcat = 138 (±2) s 1. Biophysical techniques showed M. tuberculosis DHDPS to exist as a tetramer in solution. This is consistent with the crystal structure deposited as PDB entry 1XXX. The crystal structure of M. tuberculosis DHDPS showed active-site architecture analogous to E. coli DHDPS and a dimeric variant of M. tuberculosis DHDPS was predicted to have reduced enzyme activity. A dimeric variant of M. tuberculosis DHDPS was engineered through a rationally designed mutation to analyze the effect of disrupting quaternary structure on enzyme function. A single point mutation resulted in a variant, DHDPS-A204R, with disrupted quaternary structure, as determined by analytical ultracentrifugation and gel-filtration chromatography. DHDPS-A204R was found to exist in a concentration-dependent monomer-dimer equilibrium, shifted towards dimer by the presence of pyruvate, the first substrate that binds to the enzyme. The secondary and tertiary structure of DHDPS-A204R was analogous to wild-type M. tuberculosis DHDPS as judged by circular dichroism spectroscopy and X ray crystallography, respectively. Surprisingly, this disrupted interface mutant had similar activity to the wild type enzyme, with a kcat of 119 (±6) s-1; although, the affinity for its substrates were decreased: KM(S) ASA = 1.1 (±0.1) mM, KMpyruvate = 0.33 (±0.03) mM. These results indicated that disruption of tetramer formation does not provide an alternative direction for drug design for DHDPS from M. tuberculosis. Comparison with the recently discovered dimeric DHDPS from Staphylococcus aureus shed further light on the role of quaternary structure in DHDPS. In M. tuberculosis DHDPS-A204R and the naturally dimeric enzyme, the association of monomers into the dimer involves a greater buried surface area and number of residues than found in E. coli DHDPS-L197Y. This provides a framework to discriminate which DHDPS enzymes are likely to be inactive as dimers and will direct future work targeting the dimer-dimer interface of DHDPS as an approach for drug design.

Indian Institute Of Science / Proteins associated with metabolic pathways have received considerable attention in an effort to understand the molecular details of the complex reactions catalyzed in vivo. Enzymes belonging to these pathways have also been explored as potential targets for therapeutic intervention. The L-Lysine biosynthesis pathway in particular remains an attractive target for the design of new anti-microbial compounds. The rationale for this interest stems from the finding that the pathway for Lysine biosynthesis is only present in bacteria and is absent in humans. m-DAP/L-Lysine is an essential component of the bacterial cell wall. The L-Lysine biosynthesis pathway in bacteria is fairly diverse. Three major routes for the biosynthesis of m-DAP/L-Lysine are currently known. These are the succinylase, the acetylase and the dehydrogenase pathways. The results reported in this thesis are based on enzymes involved in the L-Lysine biosynthesis pathway of a nosocomial pathogen, Staphylococcus aureus spp. COL. The structural and biochemical characterization of three enzymes, Dihydrodipicolinate synthase (DapA), Dihydrodipicolinate reductase (DapB) and Succinyl diaminopimelate desuccinylase (DapE) provide a mechanistic rationale for the activity of these proteins. These studies reveal substantial differences in the regulatory mechanisms of these enzymes that could potentially be utilized for the design of inhibitors that specifically target this biosynthesis route. This thesis is organized as follows: Chapter 1 provides an introduction to the topic of this thesis. The first part of this chapter describes the characteristic features of Staphylococcus aureus and aspects relating to the identification, pathogenesis and genomic differences in S. aureus strains. The general features of the peptidoglycan layer of the bacterial cell wall including its structure and composition are also discussed. The second part of this chapter provides an overview of the Lysine biosynthesis pathway and details of the enzymes in this pathway. Chapter 2 describes the structural and functional features of Dihydrodipicolinate synthase (DHDPS) also referred to as DapA. DHDPS catalyzes the first committed step of L-Lysine biosynthesis. Here we report the crystal structure of the native and pyruvate complexes of S. aureus DHDPS. S. aureus DHDPS is a dimer, both in solution as well as in the crystal. The functional characterization of S. aureus DHDPS revealed that this enzyme is active as a dimer. This feature distinguishes the S. aureus enzyme from the E. coli homologue where a tetrameric quaternary arrangement is essential for the activity of this protein. A comparison between the native and pyruvate-bound structures also provides a structural basis for the ping-pong reaction mechanism of this enzyme whereby the catalytic triad is drawn closer to facilitate proton transfer upon pyruvate binding. It was also noted that unlike the E. coli homologue, S. aureus DHDPS is not feedback inhibited by lysine. The lack of feedback inhibition in S. aureus DHDPS could be attributed to a unique allosteric site. The different quaternary arrangement and a distinct allosteric pocket in this enzyme thus provide a structural template for the design of specific inhibitors for this enzyme. Chapter 3 is based on preliminary studies of Dihydrodipicolinate reductase (DHDPR), encoded by the dapB gene. DHDPR catalyzes the second committed step in m-DAP/L-Lysine biosynthesis. The dapB gene encoding DHDPR was cloned and over-expressed in E. coli. Two variations of the recombinant protein were examined- one with a hexa-histidine tag at the C-terminus and the other without any tag. The recombinant DHDPR with the C-terminal hexa-histidine tag was purified by Ni-affinity chromatography and was subsequently crystallized. However, data sets collected on these crystals could not be examined further due to pronounced pseudo-translational symmetry and poor resolution. The recombinant DHDPR protein without an expression tag was purified by anion-exchange and size exclusion chromatography. Analytical gel filtration studies with recombinant DHDPR is consistent with a tetrameric quaternary arrangement of DHDPR subunits with a calculated molecular mass of 135 kDa. Diffraction data were collected to 3.3 Å resolution on crystals of apo DHDPR. These crystals belong to the C-centered monoclinic space group (C2) with unit cell parameters a = 63.17 Å, b = 78.91 Å, c = 128.38 Å and γ = 110.0°. Assuming two molecules in the asymmetric unit, the calculated Matthew’s coefficient (Vm) was 2.32 Å3 Da-1 and solvent content was 47.0 %. Molecular replacement (MR) trials with a model combining E .coli DHDPR structures (residues 1-106 of 1ARZ and 108-241 of 1DIH) as a search model resulted in a successful MR solution with two molecules in the asymmetric unit. Chapter 4 is based on the structure and regulatory mechanism of DapE also referred to as Sapep. This enzyme belongs to the M20 family of proteases and is characterized by diverse substrate specificity and multiple functional roles. These include Succinyl diaminopimelate desuccinylase, a Mn2+-dependent di-peptidase and a -lactamase. The chemical reaction involved in all these functions is broadly similar and involves amide bond hydrolysis. In an effort to understand the structure and regulatory features of this enzyme, the structure of Sapep was determined both in a Mn2+-bound form and in a metal-free (apo) form. A comparison between these structures revealed that large inter-domain movements potentially regulate the activity of this enzyme. These structures also revealed an additional regulatory mechanism wherein the inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys155 and Cys178 are not active site residues, the reduced form of this enzyme is substantially more active as a peptidase. The characterization of disulfide bond in the apo-form of the protein in solution by mass spectrometric studies and the requirement of a reducing agent for optimal catalytic activity of this protein suggests that the conformational features noted in crystal structures are also likely in solution. The structural and biochemical features of this enzyme thus provide a basis to rationalize the multiple functional roles of this protein with potential applications to MRSA-specific therapeutic strategies. Chapter 5 provides a summary of the biochemical and structural data on the three enzymes of the L-Lysine biosynthesis pathway in S. aureus spp. COL. The emphasis of the discussion in this chapter is on features that are specific to these S. aureus enzymes. The latter part of this chapter is based on the scope of future studies in this area. The appendix sections of this thesis are based on a project involving the catalytic domain of a receptor protein tyrosine phosphatase CRYP-2/cPTPRO. Appendix-I includes the structure-function analysis of the catalytic domain of CRYP-2/cPTPRO. CRYP-2/cPTPRO is a receptor protein tyrosine phosphatase (PTP) that is selectively expressed in neurons and has been implicated in axon growth and guidance. The extracellular receptor domain of this protein has eight fibronectin typeIII repeat regions while the intracellular region consists of a catalytic PTP domain. The crystal structure of CRYP-2 revealed two molecules of the catalytic domain in the asymmetric unit. The substantial buried surface area of this crystallographic oligomer suggested a homo-dimer of the catalytic domain. Solution studies however suggested that this protein is a monomer in solution based on the elution profile of CRYP-2 in a size exclusion chromatography experiment. The monomeric nature of CRYP-2 thus suggests that dimerization induced modulation of enzyme activity, such as that seen in RPTP- where a helix-turn-helix segment of one monomer blocks the active site of the other, is not possible in the case of CRYP-2. Both monomers of CRYP-2 reveal a nitrate ion bound at the active site. An advantage provided by the crystallographic dimer of CRYP-2 was that it allowed us to visualize this protein with its active site lid (WPD-loop) in both the open and closed conformations. A structural comparison of CRYP-2 with other PTP’s suggests that minor conformational rearrangements, as opposed to dimerization, could serve to regulate the activity of members of the type III family of RPTPs. Appendix-II describes a project to examine the feasibility of utilizing mesoporous matrices of alumina and silica for the controlled inhibition of enzymatic activity. In these studies, we employed bare and functionalized mesoporous alumina and MCM48 silica to deliver para-nitrocatechol sulfate (pNCS), a potent competitive PTP inhibitor. pNCS was chosen as model inhibitor because of the ease of monitoring its release using UV-Visible absorption spectroscopy. CRYP-2 was used as model enzyme in this analysis. Inhibition of catalytic activity was examined using the sustained delivery of para-nitrocatechol sulfate (pNCS) from bare and amine functionalized MCM48 and Al2O3. Among the various mesoporous matrices employed, amine functionalized MCM48 exhibited the best controlled release of pNCS and inhibition of CRYP-2. Appendix-3 incorporates additional methodologies and technical details that could not be included in the main body of the thesis.