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dc.contributor.advisorTonge, Peter Jen_US
dc.contributor.authorLi, Huei-Jiunen_US
dc.contributor.otherDepartment of Chemistryen_US
dc.date.accessioned2013-05-24T16:38:16Z
dc.date.available2013-05-24T16:38:16Z
dc.date.issued1-May-12en_US
dc.date.submitted12-Mayen_US
dc.identifierStonyBrookUniversityETDPageEmbargo_20130517082608_116839en_US
dc.identifier.urihttp://hdl.handle.net/1951/60235
dc.description232 pg.en_US
dc.description.abstractSlow-onset inhibition is observed for many successful drugs on the market presumably since this leads to a better pharmacodynamic profile. However, the structural basis for slow-onset inhibition is largely unexplored and there is no rational rule for developing such kinetic properties. In our efforts to combat M. tuberculosis through the inhibition of its FAS-II enoyl-ACP reductase InhA, we have discovered a diphenyl ether scaffold that exhibits slow-onset inhibition, and used this as a model system to uncover the nature of the slow step that occurs on the timescale of minutes and longer. Previously it was found that slow-onset inhibition correlated to the ordering of helix-6 on InhA. In the present work, from the conformational space covered by a large number of crystal structures, separate conformational states of InhA are identified and the energy barrier revealed by two-dimensional energy profiles. The size of the barrier and relative stabilities of the two major conformational states rationalize observations in kinetic experiments and crystal structures, supporting that slow-onset inhibitors initially bind with an InhA conformational state competent in binding the natural substrate, the long chain fatty acyl-carrier protein, and the complex subsequently undergoes isomerization to an otherwise naturally unfavorable conformer of InhA. Analysis of crystal structures along the binding reaction coordinate reveals that the helix-6 ordering event is the consequence of a large-scale local refolding process involving at least 30 residues induced by the interactions with the inhibitor. MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 ? structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate -proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase, is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in -proton acidity. The designated trpE gene (Rv1609) from Mycobacterium tuberculosis was expressed in E. coli and characterized. While TrpE displays normal NH4+ and Mg2+ dependence and inhibition by tryptophan, as expected for an anthranilate synthase, its ability to produce anthranilate is affected by contaminating enzymes that display chorismate mutase and prephenate dehydratase activities. Introduction of the trpG (Rv0013) gene product into the reaction mixture preferably partitions chorismate into the direction of anthranilate production. Kinetic analysis demonstrates that the improved efficiency of anthranilate production is achieved by greater affinity of TrpE for chorismate and also an increase in kcat for the reaction. Intriguingly, there is no evidence for a tight complex between TrpE and TrpG, unlike other characterized anthranilate synthases that exhibit cooperativity. Moreover, while AS activity was optimized, the reaction intermediate ADIC was found to accumulate although other anthranilate synthases are not known to accumulate ADIC. The study reveals the importance of complex formation for AS catalysis and again raises the long standing question of how anthranilate synthases catalyze pyruvate elimination.en_US
dc.description.abstractSlow-onset inhibition is observed for many successful drugs on the market presumably since this leads to a better pharmacodynamic profile. However, the structural basis for slow-onset inhibition is largely unexplored and there is no rational rule for developing such kinetic properties. In our efforts to combat M. tuberculosis through the inhibition of its FAS-II enoyl-ACP reductase InhA, we have discovered a diphenyl ether scaffold that exhibits slow-onset inhibition, and used this as a model system to uncover the nature of the slow step that occurs on the timescale of minutes and longer. Previously it was found that slow-onset inhibition correlated to the ordering of helix-6 on InhA. In the present work, from the conformational space covered by a large number of crystal structures, separate conformational states of InhA are identified and the energy barrier revealed by two-dimensional energy profiles. The size of the barrier and relative stabilities of the two major conformational states rationalize observations in kinetic experiments and crystal structures, supporting that slow-onset inhibitors initially bind with an InhA conformational state competent in binding the natural substrate, the long chain fatty acyl-carrier protein, and the complex subsequently undergoes isomerization to an otherwise naturally unfavorable conformer of InhA. Analysis of crystal structures along the binding reaction coordinate reveals that the helix-6 ordering event is the consequence of a large-scale local refolding process involving at least 30 residues induced by the interactions with the inhibitor. MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 ? structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate -proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase, is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in -proton acidity. The designated trpE gene (Rv1609) from Mycobacterium tuberculosis was expressed in E. coli and characterized. While TrpE displays normal NH4+ and Mg2+ dependence and inhibition by tryptophan, as expected for an anthranilate synthase, its ability to produce anthranilate is affected by contaminating enzymes that display chorismate mutase and prephenate dehydratase activities. Introduction of the trpG (Rv0013) gene product into the reaction mixture preferably partitions chorismate into the direction of anthranilate production. Kinetic analysis demonstrates that the improved efficiency of anthranilate production is achieved by greater affinity of TrpE for chorismate and also an increase in kcat for the reaction. Intriguingly, there is no evidence for a tight complex between TrpE and TrpG, unlike other characterized anthranilate synthases that exhibit cooperativity. Moreover, while AS activity was optimized, the reaction intermediate ADIC was found to accumulate although other anthranilate synthases are not known to accumulate ADIC. The study reveals the importance of complex formation for AS catalysis and again raises the long standing question of how anthranilate synthases catalyze pyruvate elimination.en_US
dc.description.sponsorshipStony Brook University Libraries. SBU Graduate School in Department of Chemistry. Charles Taber (Dean of Graduate School).en_US
dc.formatElectronic Resourceen_US
dc.language.isoen_USen_US
dc.publisherThe Graduate School, Stony Brook University: Stony Brook, NY.en_US
dc.subject.lcshBiochemistry--Chemistryen_US
dc.subject.otherAnthranilate synthase, Catalytic mechanism, Crystal structure, Enoyl reductase, Naphthoate synthase, Slow-onset inhibitionen_US
dc.titleStructural Mechanisms of Catalysis and Inhibition of Enzymatic Drug Targets Involved in Fatty Acids and Menaquinone Biosyntheses of Mycobacterium tuberculosisen_US
dc.typeDissertationen_US
dc.description.advisorAdvisor(s): Tonge, Peter J. Committee Member(s): Johnson, Francis ; Fowler, Frank W; Seeliger, Markus.en_US
dc.mimetypeApplication/PDFen_US
dc.embargo.releaseMay-14en_US
dc.embargo.period2 Yearsen_US


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