Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2008. / Title from second page of PDF document. Includes bibliographical references.

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19195号 / 農博第2134号 / 新制||農||1034(附属図書館) / 学位論文||H27||N4941(農学部図書室) / 32187 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 谷 史人, 教授 保川 清, 准教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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N-Hydroxylating monooxygenases (NMOs) are flavin dependent enzymes that primarily catalyze the hydroxylation of L-ornithine or L-lysine. This is the first, committed step to siderophore biosynthesis. Pathogenic microbes including Aspergillus fumigatus and Mycobacterium tuberculosis secrete these low molecular weight compounds in order to uptake FeIII from their hosts for their metabolic needs when establishing infection. Therefore, members of this family of enzymes represent novel drug targets for the development of antibiotics. Here, we present the detailed functional and structural analysis of the L-ornithine monooxygenase SidA from Aspergillus fumigatus and the L-lysine monooxygenases MbsG from Mycobacterium smegmatis and NbtG from Nocardia farcinica. The detailed chemical mechanism for flavin oxidation in SidA was elucidated for formation of the C4a-hydroperoxyflavin, deprotonation of L-ornithine, and for the chemical steps of hydrogen peroxide elimination and water elimination. This was performed through a combination of kinetic isotope effect, pH, and density functional theory studies. Also, important residues involved in substrate binding and catalysis were characterized using site-directed mutagenesis for both SidA and NbtG. These include residues involved in coenzyme selectivity, substrate binding, and residues important in C4a-hydroperoxyflavin stabilization and flavin oxidation. The kinetic mechanisms of the L-lysine monooxygenases MbsG and NbtG were characterized which show unique differences with SidA. These include differences in coenzyme selectivity, and C4a-hydroperoxyflavin stabilization. Lastly, the three-dimensional structure of NbtG was solved using X-ray crystallography which is the first structure of a lysine monooxygenase. The structure shows the NADPH-binding domain is rotated ~30° relative to the FAD-binding domain which occludes NADP+ binding in NbtG. Unlike SidA, NbtG does not stabilize a C4a-hydroperoxyflavin and this occlusion observed in the structure might explain this difference. This highlights both the structural and mechanistic diversities among NMOs and the data presented here provides valuable information for the future development of specific inhibitors of NMOs. / Ph. D.

Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH).  MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification.  Therefore, MSH is considered a potential drug target.  The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins).  In this dissertation, we present detailed functional analysis of MshB.  Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including  metal ion availability and oxidative conditions.  MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate.  Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products.  A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate.  Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB.  Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates.  Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment. / Ph. D.

The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform computational molecular docking in our efforts to develop new inhibitors against CTX-M. The goal of this project was to determine the hydrogen bonding network and proton transfer process at different stages of the reaction pathway as well as develop novel inhibitors against CTX-M β-lactamases. The results I have obtained from the project have elucidated the catalytic mechanism of CTX-M β-lactamase in unprecedented detail and facilitated the development of novel inhibitors for antibiotic drug discovery. The first aim of the project focused on developing high affinity inhibitors against class A β-lactamase using a structure-based drug discovery approach, which ultimately led to the identification of CTX-M9 inhibitors with nanomolar affinity. Compound design was based on the initial use of computational molecular docking results along with x-ray crystal structures with known inhibitors bound in the active site. In addition, chemical synthesis was used to build and extend the existing inhibitor scaffold to improve affinity to CTX-M9 and related serine β-lactamases. Through a fragment-based screening approach, we recently identified a novel non-covalent tetrazole-containing inhibitor of CTX-M. Structure-based design was used to improve the potency of the original tetrazole lead compound more than 200-fold with the use of small, targeted structural modifications. A series of compounds were used to probe specific binding hotspots present in CTX-M. The designed compounds represent the first nM-affinity non-covalent inhibitors of a class A β-lactamase. The complex structures of these potent compounds have been solved using high resolution x-ray crystallography at ~ 1.2-1.4 Å, which provides valuable insight about ligand binding and future inhibitor design against class A β-lactamases. Specifically, the first aim of the project was to use ultra-high resolution x-ray crystallography to study β-lactamase catalysis. Through the use of ultra-high resolution x-ray crystallography with non-covalent and covalent inhibitors, I was able to structurally characterize the critical stages of the enzyme mechanism. Here we report a series of ultra-high resolution x-ray crystallographic structures that reveal the proton transfer process for the early stages of the class A β-lactamase catalytic mechanism. The structures obtained include an a 0.89 Å crystal structure of CTX-M β-lactamase in complex with a recently-developed 89 nM non-covalent inhibitor, and a 0.80 Å structure in complex with an acylation transition state boronic acid inhibitor. Nearly all the hydrogen atoms in the active site, including those on the ligand, polar protein side chains and catalytic water, can be identified in the unbiased difference electron density map. Most surprisingly, compared with a previously determined 0.88 Å apo structure determined under the same conditions, the hydrogen-bonding network has undergone a series of reshuffling upon the binding of the non-covalent ligand. Two key catalytic residues, Lys73 and Glu166, appear to have both changed from a charged state to being neutral. Interestingly, structural evidence suggests the presence of a low barrier hydrogen bond (LBHB) shared between Lys73 and Ser70. These unprecedented detailed snapshots offer direct evidence that ligand binding can alter the pKa's of polar protein side chains and their affinities for protons. Such effects can be a common mechanism utilized by enzymes to facilitate the proton transfer process of a reaction pathway. They also have important implications for computational modeling of protein-ligand interactions. Ultra-high resolution x-ray crystallography allowed us to determine the hydrogen atom positions for key active site residues involved in catalysis. As a result, the ability to characterize the hydrogen bonding network led to the determination of the specific proton transfer process that occurs during the reaction stages of the CTX-M β-lactamase mechanism. Overall, the results from this project demonstrate the effectiveness of using ultra high resolution x-ray crystallography as a useful tool to study enzyme catalysis as well as develop and discover novel inhibitors.