The spermatozoan is a highly compartmentalized structure consisting of head, midpiece and tail regions. Various structural components and numerous enzymes occupy these three compartments. Evidence for the organization of glycolytic enzymes into a macroenzyme complex has previously been presented for both skeletal muscle and Escherichia coli. A multienzyme glycolytic complex would benefit the spermatozoan by increasing fructolytic efficiency by decreasing diffusion of intermediary metabolites and by directly providing ATP for sperm cell motility and other functions. Data from the present work suggests the existence of a multiprotein complex in spermatozoa. Aldolase from bovine muscle (BMA) and sperm (BSpA) have been characterized with respect to the nature of their subcellular associations. Solubilization properties of BMA and BSpA were investigated. Deoxycholate, when included in homogenization buffer, solubilized approximately 81%, of the BMA per gram of tissue. Triton X100 in combination with phosphate buffer solubilized only 10% of the total 3SpA per cell. BMA was purified to greater than 90% homogeneity by salt fractionation, gel filtration, and phosphocellulose chromatography. Aldolase from bovine muscle was a tetramer with a molecular weight per subunit of 41,000. BSpA was partially purified using the above methods. Aldolases from bovine muscle and spermatozoa were compared with regard to their activity toward FBP and F-1-P. BMA exhibited an FBP to F-1-P activity ratio of 9.62, which is typical of type A aldolases. BSpA showed an FBP to F-1-P activity ratio of 0.038 which is not characteristic of the previously identified type A, B or C aldolases. BMA and BSpA were different with respect to solubilization, purification and kinetic properties. / Master of Science

Darmstadt, Techn. Univ., Diss., 2004.

The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties.  In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.

An efficient purification procedure of Ascaris suum muscle utilizing ion exchange column chromatography has been developed.

The Morita-Baylis-Hillman (MBH) reaction is a carbon-carbon (C-C) bond forming reaction between an activated alkene and an aldehyde. It is a synthetically useful reaction due to the high atom economy and retention of multiple functional groups. Unfortunately, harsh reaction conditions are required during the MBH reaction and unpredictable product stereospecificity have hampered the widespread application of this reaction. Catalysis of the MBH reaction by enzymes has the potential to allow the reaction to occur at ambient conditions, while offering scope for improving the stereospecificity. This thesis focussed on the enzyme design of a MBH enzyme using thermostable fructose-1,6- bisphosphate (FBP) aldolases as scaffolds. These enzymes were chosen because there are common features between the aldol and MBH reactions, both making use of an enol intermediate to attack the aldehyde. In addition, aldolases typically accept a wide variety of substrates. Thermostable aldolases were selected for increased temperature tolerance creating a more desirable catalyst for industrial purposes. Thermoproteus tenax FBP aldolase (TtFBPA; WT and W144L, W144Y, K177A variants) and Staphylococcus carnosus FBP aldolase (ScFruA) were expressed and purified from E. coli. While the retro-aldol reaction catalysed by these enzymes could be easily monitored, the reverse reaction (aldol synthesis) is more difficult to quantify. Multiple methodologies for high throughput spectrophotometric detection of aldol activity were developed as a method of monitoring constructs made during directed evolution of the FBP aldolases. However, none of these proved successful in robustly determining aldol activity. The dihydroxyacetone phosphate (DHAP) mimic 1-hydroxy-3-buten-2-one phosphate (HBOP) was used to assay for MBH catalysis. While crystallographic studies with TtFBPA suggest that HBOP is bound to W144L TtFBPA in a manner compatible with the MBH reaction. NMR studies could not detect any corresponding activity. This suggests further protein engineering will be required to evolve this FBP aldolase to an MBH catalyst. In addition, our crystallographic and NMR studies with TtFBPA reveals this enzyme is capable of catalysing the formation of both FBP and tagatose-1,6-bisphosphate (TBP).Additionally, we determined the first structure of ScFruA. Interestingly, NMR experiments suggested ScFruA lacks significant control of the stereospecificity of the aldol condensation reaction and appears to catalyse the formation of FBP, TBP, xyluose-1,6- bisphosphate and psicose-1,6-bisphosphate. We conclude that while FPB aldolases could indeed provide useful scaffolds for the development of an MBH catalyst, the enzymes lack any inherent activity, necessitating the need for future creation of variants. The success of this approach will depend on the ability to screen mutant libraries for MBH product formation.

It has been suggested for many decades that the essential and ubiquitous enzyme fructose 1,6-bisphosphate aldolase (FBA) could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependent (Class II) FBA, as opposed to higher organisms which possess a Schiff-base forming, metal-independent (Class I) FBA. The purpose of this doctoral project was to purify and study the inhibition of Class II FBA from pathogenic organisms. The capacity of various thiol compounds, as well as various derivatives of the metal-chelating compound dipicolinic acid, to inhibit the purified Class II FBAs from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, and Magnaporthe grisea, was compared. The genes were subcloned in the Escherichia coli vector pT7-7 and the enzymes purified to near homogeneity, and characterized using a coupled assay. A small fed-batch fermentor was used to express the enzymes in E. coli, and yields of up to 2 grams of purified protein per liter of bacterial culture were obtained. The commercially available compound 2,3-dimercaptopropane sulfonate was found to be the most effective inhibitor against the aldolase from M. tuberculosis, with a second order binding rate constant of 500 +/- 4 M-1 s-1, which is three times and twenty times higher than the constants obtained with dipicolinic acid and EDTA, respectively. In an attempt to detect the enzyme dynamics during catalysis or inhibition, tryptophan residues were used as reporter groups and introduced by site-directed mutagenesis into the catalytic mobile loops and near the active site of the aldolases from M. tuberculosis, P. aeruginosa and B. cereus. The kinetic characterization of the mutants is described; as well as the effect of substrate binding on the steady-state and time-resolved fluorescence signals. Finally, the possibility of using the recombinant Class II FBP aldolases for industrial chemical synthesis was explored by measuring the enzymatic stability in organic solvents, at high temperatures and at different pH conditions. Surprisingly, the commercial Class I enzyme from rabbit muscle was more stable than the metalloenzymes in most conditions tested. The results presented in this thesis will be useful for the future design of Class II FBP aldolase inhibitors.

It has been suggested for many decades that the essential and ubiquitous enzyme fructose 1,6-bisphosphate aldolase (FBA) could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependent (Class II) FBA, as opposed to higher organisms which possess a Schiff-base forming, metal-independent (Class I) FBA. The purpose of this doctoral project was to purify and study the inhibition of Class II FBA from pathogenic organisms. The capacity of various thiol compounds, as well as various derivatives of the metal-chelating compound dipicolinic acid, to inhibit the purified Class II FBAs from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, and Magnaporthe grisea, was compared. The genes were subcloned in the Escherichia coli vector pT7-7 and the enzymes purified to near homogeneity, and characterized using a coupled assay. A small fed-batch fermentor was used to express the enzymes in E. coli, and yields of up to 2 grams of purified protein per liter of bacterial culture were obtained. The commercially available compound 2,3-dimercaptopropane sulfonate was found to be the most effective inhibitor against the aldolase from M. tuberculosis, with a second order binding rate constant of 500 +/- 4 M-1 s-1, which is three times and twenty times higher than the constants obtained with dipicolinic acid and EDTA, respectively. In an attempt to detect the enzyme dynamics during catalysis or inhibition, tryptophan residues were used as reporter groups and introduced by site-directed mutagenesis into the catalytic mobile loops and near the active site of the aldolases from M. tuberculosis, P. aeruginosa and B. cereus. The kinetic characterization of the mutants is described; as well as the effect of substrate binding on the steady-state and time-resolved fluorescence signals. Finally, the possibility of using the recombinant Class II FBP aldolases for industrial chemical synthesis was explored by measuring the enzymatic stability in organic solvents, at high temperatures and at different pH conditions. Surprisingly, the commercial Class I enzyme from rabbit muscle was more stable than the metalloenzymes in most conditions tested. The results presented in this thesis will be useful for the future design of Class II FBP aldolase inhibitors.

The aldolase-dehydrogenase complex catalyzes the last two steps in the microbial meta-cleavage pathway of various aromatic compounds including polychlorinated biphenyls (bph pathway) and cholesterol (hsa pathway). The aldolase, BphI, cleaves 4-hydroxy-2-oxoacids to produce pyruvate and an aldehyde. Linear aldehydes of up to six carbons long and branched isobutyraldehyde were directly channeled to the aldehyde dehydrogenase BphJ, via a molecular tunnel, with greater than 80% efficiency. The molecular tunnel is narrow in positions lined by Gly-322 and Gly-323 in the aldolase. BphI variants G322F, G322L and G323F were found to block aldehyde channeling. The replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies, thus disproving a previous hypothesis that hydrogen bonding between the Asn-170 and the nicotinamide cofactor induces the opening of the exit of the tunnel. The H20A and Y290F BphI variants displayed significantly reduced aldehyde channeling efficiencies indicating that these residues control the entry and exit of substrates and products from the aldolase reaction. The BphI reaction was activated by NADH binding to BphJ in the wild-type enzyme and channel blocked variants. Activation of BphI by BphJ N170A, N170D and I171A was decreased by ≥ 3-fold in the presence of NADH and ≥ 4.5-fold when BphJ was undergoing turnover. These results demonstrate that the dehydrogenase coordinates catalytic activity of BphI through allostery rather than through faster aldehyde release from substrate channeling. HsaF, an ortholog of BphI from Mycobacterium tuberculosis could be expressed as a soluble dimer, however HsaF was inactive in the absence of HsaG, a BphJ ortholog. Acetaldehyde and propionaldehyde were channeled directly to HsaG with similar efficiencies as in the BphI-BphJ system. The HsaF-HsaG complex was crystallized and its structure solved to a resolution of 1.93 Å. Substitution of Ser-41 in HsaG with isoleucine or aspartate resulted in about 35-fold increase in Km for CoA but only 4-fold increase in Km for dephospho-CoA, confirming its importance in interacting with the 3’- ribose phosphate of CoA. A second gene annotated as 4-hydroxy-2-oxopentanoic acid aldolase (Rv3469c) from M. tuberculosis was expressed, purified and found to possess oxaloacetate decarboxylase and not aldolase activity.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

As doenças tropicais negligenciadas, que atingem as populações mais carentes do mundo, representam em termos humanitários e socioeconômicos uma grande preocupação global. As tripanossomíases estão entre as doenças parasitárias mais importantes, e, particularmente, a tripanossomíase africana, ou doença do sono, destaca-se como uma grave condição de saúde, causada pelo parasita unicelular Trypanosoma brucei. Dentre os principais alvos metabólicos considerados para o desenvolvimento de novos fármacos para o tratamento das tripanossomíases, a glicólise recebe especial atenção em função de seu papel vital no processo de produção de ATP para o parasita que vive na corrente sanguínea. Esta tese de doutorado tem como objetivo identificar novos candidatos a inibidores da enzima aldolase (EC 4.1.2.13) da via glicolítica de T. brucei. Considerando-se que o alvo macromolecular em questão é validado para o planejamento de fármacos, inibidores desta enzima são candidatos a novos agentes quimioterápicos. Este trabalho explora a integração de métodos experimentais e computacionais através de estratégias de planejamento de fármacos baseado na estrutura do receptor (SBDD, na sigla inglesa para structure-based drug design) e na estrutura do ligante (LBDD, na sigla inglesa para ligand-based drug design) para a identificação de inibidores da enzima alvo. Foram produzidos resultados significativos, tais como a identificação através de triagens virtuais em larga escala de novas moléculas capazes de inibir a atividade da aldolase. Adicionalmente, destaca-se a obtenção de protocolos de expressão, purificação e cristalização para a enzima alvo. Como parte da estratégia de identificação de novos inibidores da aldolase, foram desenvolvidos modelos de QSAR 2D e 3D e estudos de dinâmica molecular. / Neglected tropical diseases, which affect the poorest populations across the developing world, are a major global concern. The trypanosomiases are amongst the most serious neglected tropical diseases, and particularly, African trypanosomiasis (sleeping sickness), caused by the unicellular parasite Trypanosoma brucei, appears as a fatal condition. The glycolytic pathway emerges as a promising target among the metabolic pathways for the development of new drugs, due to its essential role in the ATP generating process in the bloodstream form of the parasite. The goal of this work is to identify new inhibitors for the glycolytic enzyme aldolase (EC 4.1.2.13) from Trypanosoma brucei. Inhibitors of this enzyme are drug candidates with high potential for clinical development, as the respective target enzyme was validated as a molecular target for the therapy of trypanosomiasis. The strategy employed in this study includes the integration of SBDD (structure-based drug design) and LBDD, (ligand-based drug design) for the identification of inhibitors of the target enzyme, through the combination of computational and experimental methodologies. Significant results were obtained, such as the identification of new small molecule inhibitors of the aldolase enzyme through high-throughput virtual screening. Additionally, it is highlighted the standardization of expression, purification and crystallization protocols for the target enzyme. As a component of the strategy for the identification of novel aldolase inhibitors, 2D and 3D QSAR models were developed, as well as molecular dynamics studies.