Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 127-143). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The three-dimensional structures of biological macromolecules and molecular assemblies are becoming increasingly important with the changing methodologies of drug discovery. The structures aid in understanding of protein function at the molecular level: be it a macromolecular assembly, a cytosolic enzyme or an intermembrane receptor molecule. X-ray crystallography is the most powerful technique to obtain the three-dimensional structures of such molecules at or near atomic resolution. With such a wide-spread importance, crystallography is an integral part of structural biology and also of the current drug discovery programs. The present thesis mainly deals with application of the crystallographic techniques for understanding the structure and function of adenylosuccinate lyase (ASL) from bacterial pathogens Salmonella typhimurium and Mycobacterium tuberculosis as well as its non-pathogenic counterpart Mycobacterium smegmatis. Studies were also carried out to understand the structure-function relationship of the protease in the plant virus Sesbania Mosaic Virus (SeMV). The thesis has been divided into six chapters. The first chapter contains an introduction to nucleotide synthesis and ASL superfamily of enzymes known as the aspartase/fumarase superfamily based on the published literature. Chapter 2 provides the details of the techniques used for the investigations presented in this thesis. Chapters 3-5 deal with the structural and functional studies carried out on ASL from the three bacterial organisms. Chapter 6 deals with the simulation studies carried out on SeMV protease. Mechanism and importance of nucleotide synthesis is introduced in Chapter 1, with special emphasis on purine de novo and salvage pathways. ASL is introduced as an important enzyme for purine synthesis. Its superfamily, the aspartase/fumarase superfamily of enzymes is described in detail with respect to its structure, function and pathophysiology. Objectives of the present study are outlined towards the end of the chapter. The experimental and computational techniques utilized during the course of my research are described in Chapter 2. These techniques include gene cloning, protein expression and purification, kinetic and biophysical characterization of proteins, crystallization, X-ray diffraction, data collection and processing, structure solution, refinement, model building, validation and structural analysis, phylogenetic studies, molecular docking and molecular dynamic simulation studies. Adenylosuccinate lyase is an important enzyme participating in purine biosynthesis. With the emergence of drug resistant variants of various pathogens, ASL has been recognized as a drug target against microbial infections. Chapter 3 deals with the structural and functional characterization of ASL from Salmonella typhimurium. Two constructs of the StASL gene were cloned and expressed leading to the purification of truncated (residues 1-366) and full-length (residues 1-456) polypeptides. Crystallization of the two polypeptides resulted in three independent structures. The full-length structure was very similar to the E. coli ASL structure consistent with 95% amino acid sequence identity between the two polypeptides. However, the truncated structures showed large distortions, especially of the active site residues, accounting for the catalytic inactivity of the truncated polypeptide in spite of retaining all residues considered important for function. The full-length ASL was catalytically active. A unique feature observed in StASL, not reported in other ASLs, was its allosteric regulation by the substrate. Kinetic studies also revealed hysteretic behavior of the enzyme. The electron density map of the full-length structure showed two novel densities on the molecular 2-fold axis into each of which a molecule of cadavarine could be fitted. Docking studies revealed a ligand-binding site at the inter-subunit interface between the two observed densities which might represent a potential allosteric site. Combining the structural and kinetic results, a possible morpheein model of allosteric regulation of StASL was hypothesized. Chapter 4 deals with the crystallographic and kinetic investigations on ASL from Mycobacterium smegmatis and Mycobacterium tuberculosis. MsASL and MtbASL were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at 2.16 Å resolution. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. Most of the active site residues were found to be conserved with the exception of Ser 148 and Gly 319 of MsASL. Ser 148 is structurally equivalent to a threonine in most other ASLs. Gly 319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active when compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria, their high GC containing genomes as well as with their dependence on other salvage pathways for the supply of purine nucleotides. Chapter 5 deals with the identification of the catalytic residues important for ASL catalysis in view of the earlier conflicting reports on the identity of these residues. pH-dependent kinetic studies were performed on full-length StASL. The theory behind these studies is also described in this chapter. Two residues with pKa values of 6.6 and 7.7 were identified as essential for the enzymatic activity. These results were interpreted along with structural comparison of MsASL and other superfamily enzymes with ordered C3 loops. They suggest that His 149 and either Lys 285 or Ser 279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. The final Chapter 6 of the thesis deals with the structural and dynamic studies carried out on Sesbania mosaic virus (SeMV) protease. The chapter begins with a general introduction to viruses, followed by a brief summary of SeMV. The goal of this study is to understand the interactions between the protease and VPg at a structural level using the information available from biochemical studies. Crystallographic studies initiated for the mutant H275APro and Y315APro were unsuccessful due to the insolubility of the proteins. Co-crystallization or soaking experiments of wild type protease with cognate peptides were unsuccessful due to the inability of the enzyme to bind to its substrates in the absence of VPg. Higher resolution structure of wild type protease did not yield any new insights when compared to the earlier reported structure determined at a lower resolution. In the absence of structural insights, molecular dynamic simulations were carried out on wild type protease structure and in silico generated mutants using GROMACS package. The studies showed the importance of flipping of residue Phe 301 and opening-closing of the loop region corresponding to residues 301-308 for the catalytic mechanism. The thesis concludes with Future perspectives of the various studies carried out on ASL and SeMV protease. The atomic coordinates determined from the work presented in this thesis have been deposited in the PDB and the assigned PDB codes are reported in the respective chapters. Publications cited in the thesis are listed in the Bibliography section.

O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos. / Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.

O Schistosoma mansoni, parasita responsável pela esquistossomose (barriga dágua), doença que afeta cerca de 300 milhões de pessoas em todo mundo, não possui a via de síntese de purinas, dependendo integralmente da via de salvação de purinas para seu suprimento dessas bases. Uma vez que a terapia se resume a administração de um único fármaco, o praziquantel, diversos casos de resistência do parasita a esse medicamento foram reportadas, sendo assim esta via tem sido citada como alvo potencial para o desenvolvimento de novos fármacos contra a doença. As enzimas adenosina kinase (AK), hipoxantina-guanina fosforibosiltransferase (HGPRT), adenilsuccinato liase (ADSL) e adenilsuccinato sintetase (ADSS) são enzimas chave desta via. Este trabalho faz parte de um projeto maior que visa a obtenção de todas as estruturas das enzimas envolvidas na via de salvação de purinas de Schistosoma mansoni. O cDNA correspondente às enzimas foi amplificado e clonado no vetor de expressão pOPIN; as enzimas AK isoforma 1, HGPRT isoforma 1 e ADSL foram expressas em E. coli Lemo21(DE3) e HGPRT isoforma 3 em E. coli B834(DE3); purificadas em coluna de cobalto agarose por afinidade, concentradas e cristalizadas no kit de cristalização Morpheus (Molecular Dimensions) no Oxford Protein Production Facility (OPPF) em Harwell UK. As coletas de dados por difração de raio-X foram realizadas no Síncrotron Diamond Light Source (DLS) - UK. Foram coletadas duas estruturas de ADSL, a 2.36Å de resolução em complexo com AMP e 2.14Å na forma Apo. A análise das estruturas revelou uma estrutura tetramérica bastante conservada entre as ADSLs, sendo este estado de oligomerização requerido, uma vez que resíduos de três das quatro subunidades compõem o sítio ativo. Apesar do sítio ativo ser altamente conservado entre SmADSL e ADSL humana, a interface dimérica dessas enzimas tem se apresentado suficientemente distintas, o que pode representar um potencial alvo para o desenvolvimento de um inibidor. O ensaio de atividade enzimática de ADSL revelou uma reação endotérmica, indicando que a contribuição da entropia relacionada a grande quantidade de moléculas de água presentes no sítio ativo é importante para a reação cinética. Após diversos experimentos de otimização dos cristais de HGPRT1 e aproximadamente 200 cristais testados, foi obtida uma estrutura em complexo com IMP a 2.8Å de resolução. A análise da estrutura revelou uma estrutura tetramérica. Apesar das subunidades não compartilharem o sítio ativo, este estado de oligomerização é requerido, uma vez que resíduos que compõem o sítio ativo também estão envolvidos em interações na interface dimérica, orientando o resíduo invariável Arg206 na direção do sítio ativo. Foram identificadas quatro mutações na região do sítio ativo entre SmHGPRT e HGPRT humana: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. Desta forma, a obtenção das estruturas contribui para o entendimento bioquímico desta via essencial para o parasita e de como este pode ser seletivamente privado de recursos. / Schistosoma mansoni is the parasite responsible for schistosomiasis, disease that affects about 300 million people worldwide, and does not have the purine de novo pathway, depending entirely on the purine salvage pathway to supply its demands on purines. Currently, both direct treatment and most disease control initiatives, rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, has stimulated efforts to develop new drugs for the treatment of schistosomiasis. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenosine kinase (AK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenylosuccinate lyase (ADSL), adenylosuccinate synthetase (ADSS) are key enzymes in this pathway. This work is part of a larger project aimed at obtaining all the structures of enzymes involved in purine salvage pathway of Schistosoma mansoni. The cDNA corresponding to the enzymes was amplified and cloned in vector pOPIN, AK isoform 1, HGPRT isoform 1 and ADSL were expressed in E. coli Lemo 21 (DE3) and HGPRT isoform 3 in E. coli B834(DE3); purified in cobalt agarose column, concentrated and crystallized in several conditions of the Morpheus (Molecular Dimensions) crystallization kit at the Oxford Protein Production Facility (OPPF) in Harwell UK. The data collection by xray diffraction were performed at Diamond Light Source UK. Two ADSL structures were obtained, ADSL in complex with AMP at 2.36Å resolution and ADSL Apo form at 2.14Å The analysis revealed a tetrameric structure highly conserved between ADSLs, and this oligomerization state is required since residues three of the four subunits comprise the active site. Despite the active site being highly conserved between human ADSL and SmADSL, the dimeric interface of these enzymes it has been shown sufficiently distinct, which may represent a potential target for the development of an inhibitor. The ADSL enzymatic activity assay showed an endothermic reaction, indicating the contribution of the entropy related to the large quantity of water molecules present in the active site is important for the reaction kinetics. After several optimization experiments of HGPRT1 crystals and about 200 crystals tested was obtained a structure in complex with IMP at 2.8Å resolution. The structure analysis revealed a tetrameric structure. Despite the subunits do not share the active site, this oligomerization state is required, since residues that make up the active site are also involved in interactions in dimeric interface, guiding the invariable residue Arg206 toward the active site. Four mutations were identified in the region of the active site between SmHGPRT and human HGPRT: Ile149Met, Pro176Arg, Val189Ile e Arg192Lys. These structures increase the important structural information available about the Schistosoma mansoni purine salvage pathway and how it can be selectively private resources.

Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...