Thesis (doctoral)--Universität Hamburg, 1980.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyses the first step in purine salvage. A complete deficiency of the enzyme results in the devastating neurological symptoms of the Lesch-Nyhan syndrome. The Lesch-Nyhan syndrome is characterised by purine overproduction leading to, hyperuricemia and gout and a central nervous system disorder characterised by severe, spasticity, choreoathetosis, mental retardation and compulsive self-mutilatory behaviour, A partial deficiency of the enzyme results in purine overproduction, gout and occasionally, mild neurological symptoms. Patients are spared the compulsive self-mutilation of the Lesch-Nyhan syndrome. The major part of the thesis consists of the characterisation of the molecular defects in nine patients with the Lesch-Nyhan syndrome. The polymerase chain reaction was used to amplify reverse transcribed HPRT mRNA. The coding region of the amplified HPRT cDNA was either directly sequenced, or cloned and sequenced. All the mutations characterised were insertion or deletion events which resulted in premature termination of the predicted protein. Three patients were found to have a deletion of exon 7, two patients had single base insertions, while two patients appeared to have a complete deletion of the HPRT gene. An insertion in one patient was the result of a mutation within. intron 6 which created a new splice donor site. The new splice donor site in concert with a cryptic splice acceptor resulted in the creation of a new exon. A deletion of exons 2, 3 and 4 in another patient was found to lead to the alternative splicing of exon 5. These unusual splice junction mutations provided in viva support for the exon definition model of pre-mRNA splicing.

The patient (T.K.), was first diagnosed as having a partial hypoxanthine-guanine phosphoribosyltransferase deficiency in 1978 when he was 20 years old. At presentation, he complained of a colicky loin-pain which radiated into his groin, and that he had had dark urine for a month. He was shown to have haematuria and urate crystalluria, and had a serum urate of 0.8mmol/l (reference range 0.12-0.5mmol/l). The diagnosis was confirmed by demonstrating a haemolysate hypoxanthine-guanine phosphoribosyltransferase activity of 550μU/mg Hb (reference range 1680-2480μU/mg Hb). Studies to determine whether the low hypoxanthine-guanine phosphoribosyltransferase activity was caused by an altered Kₘ of the enzyme for one of its substrates, showed that there was substrate inhibition of the enzyme activity by hypoxanthine. This thesis examines the patient and the variant HPRT at three levels. Firstly, a detailed and comprehensive study of the kinetic properties of the variant enzyme was made. The novel feature of the kinetics is the presence of substrate inhibition by the purine bases, with a true Kᵢ value for hypoxanthine of 80± 20μM, and a normal value for the true Kₘ. The pattern of substrate inhibition is characteristic of that associated with the formation of a dead-end complex and double inhibition experiments indicate that the form of this complex is enzyme-hypoxanthine-PPi. These unusual kinetic properties provided an opportunity to study the order of substrate binding in a way not possible for the normal enzyme and showed an ordered sequential reaction mechanism. Some limited protein-structural studies were performed and showed an altered electrophoretic mobility for the variant enzyme in non-denaturing gels. Secondly, the purine metabolic pathways in cultured cells, derived from T.K., from a patient with the Lesch-Nyhan syndrome, and from normal individuals, were studied. The cells were labelled with precursors of the de novo or of the salvage pathways, usually in the presence of a reference label, and sometimes in the presence of inhibitors of the various steps in the purine metabolic pathways. Hypoxanthine salvage was about 10% of that of control cultures. The growth of cells in a variety of selective media was also studied. Thirdly, as physician in charge of T.K., I could monitor the progress of his hyperuricaemia and observe the effects of therapy throughout the duration of this project.

A 2,8-DHA Urolitíase é uma doença resultante de uma desordem hereditária que leva a deficiência de atividade da enzima APRT do grupo das PRTases. Até o momento, foram encontradas 18 mutações em pacientes, das quais 7 são missense. O presente trabalho dedica-se ao estudo funcional e estrutural dessas 7 mutações e da deleção ΔF173. Construções dos mutantes D65V, L110P, M136T, R67Q, R89Q, I112F e F173G foram obtidas no vetor pET-29a(+) e clonados em E. coli. Os protocolos de expressão e purificação foram estabelecidos, onde as enzimas foram obtidas após uma etapa cromatográfica na coluna CHTTM Hidroxiapatita Cerâmica no grau de pureza necessário. A única exceção foi o mutante I112F que se mostrou insolúvel durante a expressão. Um ensaio cinético acoplado através de kit para detecção de PPi foi padronizado e permitiu a caracterização cinética da hAPRT nativa e de seu mutantes. Os mutantes apresentaram perdas de eficiência de uma ordem de magnitude, com exceção do F173G, cujos valores foram comparáveis aos da hAPRT nativa. Ensaios de cristalização foram realizados para todos os mutantes e resultaram em cristais para os mutantes F173G, R67Q e R89Q. Esses cristais foram coletados na linha de Cristalografia de Macromoléculas 2 no LNLS. Os conjuntos de dados estão sendo analisados e as estruturas estão em fase final de refinamento. / 2,8-DHA Urolithiasis is a disease resulting from an inherited disorder that leads to deficiency of APRT, an enzyme of the PRTases group. So far, 18 mutations were found in patients, 7 of which are missense. This work is dedicated to the functional and structural study of these 7 mutations and the deletion ΔF173. Mutants constructions D65V, L110P, M136T, R67Q, R89Q, I112F and F173G were obtained in the vector pET-29a (+) and cloned in E. coli. The expression and purification protocols were established and the enzymes were obtained after 1 chromatographic step using the CHTTM Hydroxyapatite Ceramics column in the required purity. The only exception was the mutant I112F that resulted insoluble during expression. A coupled kinetic assay using PPi detection kit was standardized and allowed the kinetic characterization of native hAPRT and its mutants. The mutants had an efficiency loss of an order of magnitude, with the exception of F173G, whose values were comparable to those of native hAPRT. Crystallization trials were performed for all mutants and resulted in crystals for mutants F173G, R67Q and R89Q. These crystals were collected on Macromolecules Crystallography 2 line at LNLS. The datasets are being analyzed and the structures are in the final stages of refinement.

A 2,8-DHA Urolitíase é uma doença resultante de uma desordem hereditária que leva a deficiência de atividade da enzima APRT do grupo das PRTases. Até o momento, foram encontradas 18 mutações em pacientes, das quais 7 são missense. O presente trabalho dedica-se ao estudo funcional e estrutural dessas 7 mutações e da deleção ΔF173. Construções dos mutantes D65V, L110P, M136T, R67Q, R89Q, I112F e F173G foram obtidas no vetor pET-29a(+) e clonados em E. coli. Os protocolos de expressão e purificação foram estabelecidos, onde as enzimas foram obtidas após uma etapa cromatográfica na coluna CHTTM Hidroxiapatita Cerâmica no grau de pureza necessário. A única exceção foi o mutante I112F que se mostrou insolúvel durante a expressão. Um ensaio cinético acoplado através de kit para detecção de PPi foi padronizado e permitiu a caracterização cinética da hAPRT nativa e de seu mutantes. Os mutantes apresentaram perdas de eficiência de uma ordem de magnitude, com exceção do F173G, cujos valores foram comparáveis aos da hAPRT nativa. Ensaios de cristalização foram realizados para todos os mutantes e resultaram em cristais para os mutantes F173G, R67Q e R89Q. Esses cristais foram coletados na linha de Cristalografia de Macromoléculas 2 no LNLS. Os conjuntos de dados estão sendo analisados e as estruturas estão em fase final de refinamento. / 2,8-DHA Urolithiasis is a disease resulting from an inherited disorder that leads to deficiency of APRT, an enzyme of the PRTases group. So far, 18 mutations were found in patients, 7 of which are missense. This work is dedicated to the functional and structural study of these 7 mutations and the deletion ΔF173. Mutants constructions D65V, L110P, M136T, R67Q, R89Q, I112F and F173G were obtained in the vector pET-29a (+) and cloned in E. coli. The expression and purification protocols were established and the enzymes were obtained after 1 chromatographic step using the CHTTM Hydroxyapatite Ceramics column in the required purity. The only exception was the mutant I112F that resulted insoluble during expression. A coupled kinetic assay using PPi detection kit was standardized and allowed the kinetic characterization of native hAPRT and its mutants. The mutants had an efficiency loss of an order of magnitude, with the exception of F173G, whose values were comparable to those of native hAPRT. Crystallization trials were performed for all mutants and resulted in crystals for mutants F173G, R67Q and R89Q. These crystals were collected on Macromolecules Crystallography 2 line at LNLS. The datasets are being analyzed and the structures are in the final stages of refinement.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves 120-139).

Helicobacter pylori infects more than half of the global population and causes gastric disorders. The increasing development of antibiotic resistance by the bacterium continues to limit treatment options. The identification and characterisation of novel therapeutic targets are necessary for successful future treatment of the infection. One potential target for therapeutic intervention is the gpt gene encoded by hp0735 (jhp0672) in H. pylori strain 26695 (J99). This gene produces a putative xanthine-guanine phosphoribosyltransferase (XGPRTase), an enzyme of the purine salvage synthesis pathway. This project employed theoretical, molecular and biochemical approaches to investigate features of H. pylori gpt and XGPRTase that will serve to ascertain their therapeutic potential. The production of a functional XGPRTase by H. pylori was investigated in cell-free extracts, and the kinetic parameters of this activity were compared to those of purified rXGPRTase enzyme. The three 6-oxopurine substrates were recognised by rXGPRTase and allosteric kinetics were observed for some substrates of the enzyme in cell-free extracts and for purified enzyme. These observations indicate complex regulation and an influence of cellular interactions on activity. Bioinformatics were employed to analyse XGPRTase phylogeny, and threading techniques used to build a structural model of XGPRTase. The enzyme is significantly divergent from the equivalent mammalian enzyme, and modelling identified specific features of the enzyme. Molecular approaches were utilised to analyse the essential role of gpt in H. pylori survival. These included insertional inactivation of the gpt in wild-type H. pylori strains and in mutants possessing a complementing copy of the gene present at the rdxA locus. No mutants were recovered with inactivated gpt possibly as a result of pleiotropic effects. Plasmid-mediated complementation was attempted employing IPTG-inducible shuttle vectors and did not yield any mutants. Further characterisation of H. pylori XGPRTase was performed by determining the effects of nucleotide monophosphates and purine analogues on enzyme activity. Inhibition by GMP was observed in all cases, however differences in the inhibition by other nucleotide monophosphates were found between cell-free extracts and the recombinant enzyme. Inhibition of rXGPRTase activity was observed by the purine analogue 6-mercaptopurine ribose, a compound that previously has been shown to inhibit H. pylori growth in culture.

Helicobacter pylori infects more than half of the global population and causes gastric disorders. The increasing development of antibiotic resistance by the bacterium continues to limit treatment options. The identification and characterisation of novel therapeutic targets are necessary for successful future treatment of the infection. One potential target for therapeutic intervention is the gpt gene encoded by hp0735 (jhp0672) in H. pylori strain 26695 (J99). This gene produces a putative xanthine-guanine phosphoribosyltransferase (XGPRTase), an enzyme of the purine salvage synthesis pathway. This project employed theoretical, molecular and biochemical approaches to investigate features of H. pylori gpt and XGPRTase that will serve to ascertain their therapeutic potential. The production of a functional XGPRTase by H. pylori was investigated in cell-free extracts, and the kinetic parameters of this activity were compared to those of purified rXGPRTase enzyme. The three 6-oxopurine substrates were recognised by rXGPRTase and allosteric kinetics were observed for some substrates of the enzyme in cell-free extracts and for purified enzyme. These observations indicate complex regulation and an influence of cellular interactions on activity. Bioinformatics were employed to analyse XGPRTase phylogeny, and threading techniques used to build a structural model of XGPRTase. The enzyme is significantly divergent from the equivalent mammalian enzyme, and modelling identified specific features of the enzyme. Molecular approaches were utilised to analyse the essential role of gpt in H. pylori survival. These included insertional inactivation of the gpt in wild-type H. pylori strains and in mutants possessing a complementing copy of the gene present at the rdxA locus. No mutants were recovered with inactivated gpt possibly as a result of pleiotropic effects. Plasmid-mediated complementation was attempted employing IPTG-inducible shuttle vectors and did not yield any mutants. Further characterisation of H. pylori XGPRTase was performed by determining the effects of nucleotide monophosphates and purine analogues on enzyme activity. Inhibition by GMP was observed in all cases, however differences in the inhibition by other nucleotide monophosphates were found between cell-free extracts and the recombinant enzyme. Inhibition of rXGPRTase activity was observed by the purine analogue 6-mercaptopurine ribose, a compound that previously has been shown to inhibit H. pylori growth in culture.

A Adenina Fosforibosiltransferase (APRT E.C. 2.4.2.7) pertence à família de enzimas Fosforibosil Transferases (PRTase) do Tipo I , que catalisa a conversão reversível de Adenina e 5-fosfo-α-D-ribose-1-difosfato (PRPP) em difosfato e adenosina monofosfato, um importante precursor energético da célula. A APRT integra a via de salvação de purinas, única forma de suprir o balanço de purinas em Schistosoma mansoni. Este trabalho apresenta o isolamento, clonagem, expressão heteróloga e purificação da APRT de S. mansoni a fim de caracterizá-la quanto seus parâmetros físico-químicos. Não se obtendo cristais de proteína, foram elaborados modelos tridimensionais por homologia para estudos de dinâmica molecular e avaliação conformacional via tCONCOORD. A estrutura de APRT humana foi usada como controle nas simulações. Os dados computacionais e de biologia molecular foram comparados entre si para validação mútua e, verificou-se que a análise cuidadosa de dados computacionais é capaz de fornecer informações críticas sobre a APRT, auxiliando e guiando os estudos experimentais. Ainda, as simulações de dinâmica molecular foram capazes de evidenciar a abertura de loops do sítio ativo, explicitar a importância da análise de rotâmeros em modelos, permitindo, então, rearranjar da forma correta resíduos erroneamente modelados. Por fim, um estudo envolvendo mecanismo catalítico sugere a participação de uma molécula de água abstraindo o próton ligado ao N9 da adenina e, para efeito de comparação, um mecanismo alternativo independente desta participação também foi descrito. Ambas as observações expandem a corrente visão sobre o mecanismo catalítico de APRTs. / The Adenine Phoshoribosyltransferase (APRT E.C.2.4.2.7) belongs to the Phosphoribosyltransferase enzyme family (PRTase) of type I which catalyzes the reversible conversion of adenine and 5-phospho-α-D-ribose-1-diphosphate (PRPP) in diphosphate and adenosine monophosphate, an important source of this compound for the cell. APRT is involved in the purine salvage pathway, the only way that Schistosoma mansoni has to supply its purine demands. This work shows the isolation, cloning, heterologous expression and the purification of APRT from S. mansoni in order to establish its physical chemical and kinetics parameters. Since crystallographic structure was not obtained, we built homology models to perform molecular dynamics experiments and conformational evaluation via tCONCOORD. The human APRT structure was used as control in the simulation experiments. The computational and molecular biology data were compared for consistency and the detailed analysis of the former allowed us to improve experimental manipulation of APRT. The molecular dynamics experiments showed the opening of the loops of the catalytic binding site and the key function of rotamers. Finally, we were able to suggest that a water molecule may catch the proton of adenine N9. Both observations improve the current view of APRT catalytic mechanism.

A Adenina Fosforibosiltransferase (APRT E.C. 2.4.2.7) pertence à família de enzimas Fosforibosil Transferases (PRTase) do Tipo I , que catalisa a conversão reversível de Adenina e 5-fosfo-α-D-ribose-1-difosfato (PRPP) em difosfato e adenosina monofosfato, um importante precursor energético da célula. A APRT integra a via de salvação de purinas, única forma de suprir o balanço de purinas em Schistosoma mansoni. Este trabalho apresenta o isolamento, clonagem, expressão heteróloga e purificação da APRT de S. mansoni a fim de caracterizá-la quanto seus parâmetros físico-químicos. Não se obtendo cristais de proteína, foram elaborados modelos tridimensionais por homologia para estudos de dinâmica molecular e avaliação conformacional via tCONCOORD. A estrutura de APRT humana foi usada como controle nas simulações. Os dados computacionais e de biologia molecular foram comparados entre si para validação mútua e, verificou-se que a análise cuidadosa de dados computacionais é capaz de fornecer informações críticas sobre a APRT, auxiliando e guiando os estudos experimentais. Ainda, as simulações de dinâmica molecular foram capazes de evidenciar a abertura de loops do sítio ativo, explicitar a importância da análise de rotâmeros em modelos, permitindo, então, rearranjar da forma correta resíduos erroneamente modelados. Por fim, um estudo envolvendo mecanismo catalítico sugere a participação de uma molécula de água abstraindo o próton ligado ao N9 da adenina e, para efeito de comparação, um mecanismo alternativo independente desta participação também foi descrito. Ambas as observações expandem a corrente visão sobre o mecanismo catalítico de APRTs. / The Adenine Phoshoribosyltransferase (APRT E.C.2.4.2.7) belongs to the Phosphoribosyltransferase enzyme family (PRTase) of type I which catalyzes the reversible conversion of adenine and 5-phospho-α-D-ribose-1-diphosphate (PRPP) in diphosphate and adenosine monophosphate, an important source of this compound for the cell. APRT is involved in the purine salvage pathway, the only way that Schistosoma mansoni has to supply its purine demands. This work shows the isolation, cloning, heterologous expression and the purification of APRT from S. mansoni in order to establish its physical chemical and kinetics parameters. Since crystallographic structure was not obtained, we built homology models to perform molecular dynamics experiments and conformational evaluation via tCONCOORD. The human APRT structure was used as control in the simulation experiments. The computational and molecular biology data were compared for consistency and the detailed analysis of the former allowed us to improve experimental manipulation of APRT. The molecular dynamics experiments showed the opening of the loops of the catalytic binding site and the key function of rotamers. Finally, we were able to suggest that a water molecule may catch the proton of adenine N9. Both observations improve the current view of APRT catalytic mechanism.