Understanding how protein sequence, structure and function coevolve is at the core of functional genome annotation and protein engineering. The fundamental problem is to determine whether sequence variation contributes to functional differences or if it is a consequence of evolutionary divergence that is unrelated to functional specificity. To address this problem, we cannot merely analyze sequence variation between homologous proteins that have different functions. For comparison, we need to understand the factors that determine sequence variation in proteins that have the same function, such as a set of orthologous enzymes. Here, we address this problem by analyzing the evolution of functionally important residues in the o-succinylbenzoate synthase (OSBS) family. The OSBS family consists of several hundred enzymes that catalyze a step in menaquinone (Vit. K2) synthesis. Based on phylogeny, the OSBS family can be divided into eight major subfamilies. We assayed wild-type OSBS enzyme activities. The results show that the enzymes from γ-Proteobacteria subfamily 1 and Bacteroidetes have relatively low values, the enzyme from Cyanobacteria subfamily 1 is intermediate, and the values for the proteins from the Actinobacteria and Firmicutes subfamilies are relatively high. We are using computational and experimental methods to identify functionally important amino acids in each subfamily. Our data suggest that each subfamily has a different set of functionally important residues, even though the enzymes catalyze the same reaction. These differences may have accumulated because different mutations were required in each subfamily to compensate for deleterious mutations or to adapt to changing environments. We assessed the roles of these amino acids in enzyme structure and function. Our method achieved 70% successful rate to identify positions that play important roles in one family but not another. The residues P119 and A329 play important role in D. psychrophila but not in T.fusca OSBS. We also observed two class switch mutations in T.fusca, P11 and P22. The mutations at these two position have a similar kinetic parameters as wild-type D. psychrophila OSBS.

Significant advancements in the field of membrane protein crystallography have provided in recent years invaluable images of transporter structures. These structures, however, are static and require complementary kinetic insight to understand how their mechanisms work. Electrophysiological studies of transporters permit the high quality kinetic measurements desired, but there are significant difficulties involved in analyzing and interpreting the data. Current methods allow a variety of kinetic parameters to be measured but there is a disconnect between these parameters and a fundamental understanding of the carrier. The intent of this research was to contribute new tools for studying the electrogenic kinetics of membrane transport proteins, to understand the link between these kinetics and the carrier, and to ultimately understand the mechanisms involved in transport. In this vein, two projects are explored covering two important kinetic time domains, transient and steady-state. The transient project studies the conformational changes of the unloaded carrier of SGLT1 through a multi-exponential analysis of the transient currents. Crystal structures have potentially identified a gated rocker-switch mechanism and the transient kinetics are used to support and study this kinetically. A protocol taking advantage of multiple holding potentials is used to measure the decay time constants and charge movements for voltage jumps from both hyperpolarizing and depolarizing directions. These directional measurements provide insight into the arrangement of the observed transitions through directional inequalities in charge movement, by considering the potential for a slow transition to hide a faster one. Ultimately, four carrier decays are observed that align with the gated rocker-switch mechanism and can be associated one-to-one with the movement of a gate and pore on each side of the membrane. The steady-state project considers a general theoretical model of transporter cycling. Recursive patterns are identified in the steady-state velocity equation that lead to a broad understanding of its geometric properties as a function of voltage and substrate concentration. This results in a simple phenomenological method for characterizing the I–V curves and for measuring the kinetics of rate limiting patterns in the loop, which we find are the basic structures revealed by the steady-state velocity.

Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (F6P), binds along the other dimer-dimer interface. The different functional forms BsPFK can take when in the presence of F6P and PEP can be described by the following diproportionation equilibrium: XE + EA <--> XEA + E where XE is the enzyme bound to PEP, EA is the enzyme bound to F6P, E represents the apo enzyme, and XEA is the ternary complex formed when both substrate and inhibitor are bound. Currently in the Protein Data Bank (PDB) there are two relevant forms of wild-type BsPFK, the EA form and the X'E form, which represents the enzyme bound to the PEP analog, phosphoglycolate (PGA). When comparing the EA and the X'E structures, a 7° rotation about the substrate-binding interface is observed and is termed the quaternary shift. The current study uses methyl TROSY NMR to examine the different liganded states of BsPFK, and for the first time structural data for the XEA species is shown. In addition, crystallography was used to obtain the first apo structure of BsPFK. To distinguish between changes associated with the quaternary shift and those associated with the intra-subunit tertiary changes, the variant D12A BsPFK was studied using kinetics, crystallography, and NMR. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the X'E form of wild-type. Kinetic studies of D12A BsPFK, when compared to wild-type, show a 50-fold diminished F6P binding affinity, 100-fold enhanced binding affinity, and a similar coupling constant. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the EA form of BsPFK. The variant T156A BsPFK shows similar binding, coupling, and structural characteristics to D12A BsPFK. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition.

Following conjugation with glutathione, xenobiotics are converted into cysteinylglycine conjugates, cysteine conjugates, and, finally, mercapturic acids. The structural factors determining the activities of dipeptidases for the metabolism of toxicologically-relevant cysteinylglycine conjugates are not well understood. I purified porcine kidney cortex membrane dipeptidase (MDP) to homogeneity, via phosphatidylinositol-specific phospholipase C-mediated cleavage of the protein’s membrane anchor and cilastatin affinity chromatography. The homodimeric structure of the MDP protein was confirmed by mass spectrometry. To test the enzyme activity of purified MDP, the cysteinylglycine conjugates of 1-(chloromethyl)naphthalene, 4-nitrobenzyl chloride, and 1-chloro-2,4-dinitrobenzene were synthesized and HPLC separation methods for their quantitation were developed. MDP catalyzed the hydrolysis of all three conjugates, but the rate of this activity was strongly dependent on the nature of the substituent on the cysteine sulfur atom.

Pathogenic bacteria use an arsenal of toxic protein virulence factors to cause disease in host cells. The mono-ADP-ribosyltransferase (mART) toxins are a family of exotoxins produced by pathogens which contribute to a wide range of diseases including cholera, diphtheria and whooping cough. Specifically, mART toxins act by transferring ADP-ribose from NAD+ to target proteins in host cells, altering or inhibiting target activity with deleterious downstream effects. Recently, in silico analyses have revealed two novel mARTs, Photox and Certhrax, from pathogenic organisms. Photox, from Photorhabdus luminescens was successfully expressed and purified from E. coli and was shown to target actin, specifically at Arg177. This covalent modification inhibits actin polymerization and leads to observed cytotoxicity in yeast cells. Photox has 35% identity with SpvB from Salmonella enterica, which allowed for a structural model to be built, showing the location of all characteristic mART active site components, and the binding site for potential inhibitors. Certhrax originates from Bacillus cereus G9241, implicated in a number of severe pneumonia cases. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we demonstrated that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. In vivo tests employing toxin gene expression in yeast, and receptor-mediated infection of mammalian, cells showed the extreme cytotoxicity of Certhrax (LD50 = 100 pg/mL against mouse macrophage cells), making it 60 times more toxic than its infamous counterpart, anthrax lethal factor. In vitro analysis indicated that Certhrax possesses NAD+ glycohydrolase activity, characteristic of many mART toxins, but we continue to search for the natural host protein target of this toxic enzyme. We determined the crystal structure of Certhrax to 2.2 Å, which illustrates a close structural similarity with anthrax lethal factor. Furthermore, we identified several small molecule inhibitors which show protection against Certhrax both in vitro and in vivo. We determined a 1.9 Å crystal structure of one inhibitor in complex with Certhrax. Through identification and characterization of novel mART enzymes, we seek to better understand this family of toxic enzymes to aid in the discovery and development of more potent therapeutics. / National Sciences and Engineering Research Council, Canadian Institutes of Health Research

Protein farnesylation is an essential post-translational modification required for the function of numerous cellular proteins including the oncoprotein Ras. The farnesyl transferase (FTase) catalyzed reaction is unique because farnesyl diphosphate (FPP), the farnesyl group donor for the reaction, forms a significant portion of a target protein binding site. The major goal of this research was to exploit this unique property of the FTase reaction and determine if changing the structure of the farnesyl donor group would affect FTase protein targeting. A small library of structural analogues of FPP was synthesized. Michelis-Menten steady-state kinetic analyses and competition reactions were used to determine the effect of these structural modifications on FTase targeting. We found that the analogues did affect FTase protein selectivity and that this could be exploited to induce unnatural target selectivity into the enzyme. The second goal of this research was to determine the effect of FPP analogues on the function of FTase target proteins. To test the effect of these analogues we determined whether the unnatural lipid could ablate oncogenic H-Ras biological function in a Xenopus laevis model system. Several analogues were able to disrupt oncogenic H-Ras function while others mimicked the activity of FPP. These results indicated that some of the FPP analogues may act a prenyl group function inhibitors that could lead to an important new class of anti-cancer therapeutics. Another major goal of this research was to use the FPP analogues as unnatural probes for the endogenous cellular activity of FTase target proteins. We developed antibodies to two of the unnatural FPP analogues to study their activity in cell cultureUtilizing these antibodies we found that alcohol prodrugs of the FPP analogues could be incorporated into cellular proteins in an FTase dependent manner. The ability of cell permeant analogues to be incorporated into live cells enhances the chances that such a molecule could be used to modify oncogenic cellular proteins with a prenyl group function inhibitor.

A quantitative understanding of metabolic control depends on a knowledge of the enzymes involved. The extrapolation of studies in vitro to the intact cell is controversial because the intracellular environment is relatively poorly characterised, particularly with respect to the interactions between weakly-associated enzymes. There is a clear need to study enzymes directly in the cell, yet there are few suitable techniques. Metabolites have been very successfully studied in cells by the non-invasive technique of nuclear magnetic resonance (NMR). NMR studies of enzymes in the cell have, however, been prevented by difficulties in assigning the resonances from the many proteins within the cell. A method for studying a specific enzyme in the cell has been developed, using Saccharomyces cerevisiae and phosphoglycerate kinase (PGK) as a model system. Using an inducible expression system, PGK was synthesised in the cell without significant synthesis of other proteins. With 5-fluorotryptophan in the growth medium, fluorine-labelled PGK was formed in situ. Fluorine is an excellent label for NMR since it is absent from most cells and has a high receptivity to NMR detection. ¹⁹ F NMR was used to study PGK in the intact cell. Comparisons with measurements in vitro showed that PGK was exposed to only a small fraction of the total intracellular [ADP], implying some form of compartmentalisation. The NMR relaxation properties observed in vivo and in vitro were compared with theoretical predictions. This showed that PGK was not part of a complex in the cell and that the viscosity of the cytoplasm, relative to water, was c. 4 at 30 °C. Fluorine-labelled pyruvate kinase and hexokinase have also been prepared; the spectra of these proteins in vitro are responsive to their ligands, and further work will study these proteins in vivo. NMR techniques were also applied to study the kinetics of PGK in the cell. PGK and GAPDH catalyse an ATP↔P_i exchange which is near-equilibrium in wild-type cells. ³¹P magnetisation transfer experiments in genetically manipulated cells showed that the reaction becomes unidirectional if the PGK activity is reduced by 95 %. Net flux is reduced by less than 30 %. In low-PGK cells, the ATP↔P_i exchange from oxidative phosphorylation can be isolated from that of glycolysis, facilitating direct measurements of the P:O ratio. In the cells studied, the P:O ratio was 2 to 3.

Sulfite oxidising enzymes are found in all forms of life and play an important role in detoxification of sulfite produced through biochemical processes. All known sulfite oxidising enzymes share a common molybdenum active site. The sulfite dehydrogenase (SDH) from the soil bacterium Starkeya novella differs from the vertebrate sulfite oxidases (SO) in that the heme and Mo subunits are tightly associated rather than connected by a flexible hinge. This structural integrity makes SDH an ideal model enzyme for the study of enzymatic sulfite oxidation without the complications of structural changes underlying catalysis. In human sulfite oxidase (HSO) the substitution of a conserved active site amino acid residue, Arg-160 for Gln, results in a lethal disease. A number of independent studies have been carried out in order to understand the effects of this substitution on catalysis in both human (HSO) and chicken sulfite oxidising enzymes (CSO). The focus of this work is the analogous residue in SDH, Arg 55. A number of active site substitutions have been investigated, including SDHR55Q, an analogous substitution to the lethal mutation identified in humans. In addition, the properties of the Arg residue have also been probed using a substitution to a hydrophobic residue, Met (SDHR55M) and a substitution to the positively charged Lys (SDHR55K). A fourth active site substitution, SDHH57A, was also investigated as the crystal structure of this variant indicated that His-57 plays a role in stabilising the position of Arg-55 in SDH. It was of interest to determine the effect of the instability in the position of Arg-55 on the catalytic parameters of the SDHH57A. The kinetic properties of the substituted enzymes were investigated using steady-state assays with cytochrome c as an electron acceptor. When the positive charge was lost in the case of SDHR55M and SDHR55Q, a dramatic increase in the KM (sulfite - app) of 2 – 3 orders of magnitude resulted. This indicates that the positive charge on Arg-55 is important for substrate binding. All the Arg-55 variants studied were found to have lower turnover numbers than the wild type, in particular, SDHR55Q was found to have a reduced kcat (108 s-1 vs 345 s-1 for SDHWT at pH 8). The changes in the Mo centre underlying the altered kinetic properties were investigated in detail using EPR spectroscopy of the intermediate MoV oxidation state in SDHR55Q and SDHH57A. Similar to what has been noted for HSOR160Q, a sulfate blocked form was observed at pH 6 using pulsed EPR experiments, suggesting that this substitution causes an inhibition of the hydrolysis step required to release the reaction product, sulfate. This could be a further reason for the poor catalytic activity of SDHR55Q, in particular, a reason for the low turnover rate of this variant. Unlike what was noted in HSOR160Q, where the substituted enzyme showed a dramatic decrease in rate of intramolecular transfer by three orders of magnitude compared to HSOWT, the rate of electron transfer was found to be 3 times faster in SDHR55Q relative to the wild type enzyme. These results indicate that Arg-55 is not involved in the pathway of electron transfer between the Mo and heme centres, but rather assists with the the docking of the heme group in HSO. As this process is not required in SDH, our results suggest that intramolecular electron transfer (IET) in HSOR160Q decreases because it is crucial for docking of the heme domain. Through potentiometric redox titrations, the effect of the active site amino acid substitutions on both the Mo and Fe redox potentials was investigated. No significant change was determined for the MoVI/V redox potentials, however, the heme potentials for SDHWT and SDHR55K were 40 mV higher than those of the other variants, with the lowest potentials belonging to SDHR55M and SDHH57A. Of further interest was that the MoVI/V couple is significantly lower than the heme couple (175 mV vs 240 mV respectively) in SDHWT. It appears that the positive charge of the Arg is important in regulating the heme redox potentials and could thereby contribute to modulating enzymatic activity. When SDH was immobilised on a modified pyrolytic graphite electrode, stable and high catalytic currents were observed, indicating facile heterogeneous electron transfer between the enzyme and the electrode. This good electron transfer allowed the catalytic properties of SDH and its substituted enzymes to be investigated as a function of potential. A pH dependence ( 59 mV/pH) in the catalytic operating potential was noted for SDHWT and SDHR55K, which appears to follow the pH dependence of the MoVI/V couple. This catalytic potential is pH-independent in the R55M and H57A variants, where the catalytic operating potentials appeared to follow the FeIII/II redox couple. It is proposed that two distinct pathways of electron transfer from the Mo centre to the electrode are likely to exist. The first is direct transfer from the Mo centre to the electrode at lower potential (~ 175 mV) while the second proceeds via the heme group (320 mV). The pathway followed is determined by the oxidation state of the heme group. A slight difference in the electron transfer rates of these two processes was seen, with direct transfer (from Mo) being the faster, which accounts for the unusual peak shape noted in the voltammogram for SDHWT at high sulfite concentrations, where the rate of catalytic activity slows at a higher potentials despite the greater thermodynamic driving force. This work provides new insights into the mechanism of enzymatic sulfite oxidation. Arg-55 has been shown to play an important role in the catalytic functioning of SDH in both substrate affinity and product release. Unlike what has been previously proposed, Arg-55 does not play a part in the pathway of electron transfer, but is rather involved in the regulation of the redox potentials of the metal centres in the enzyme.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2007. / Title from second page of PDF document. Includes bibliographical references.

Submitted by Emily Colferai Bonfá null (miemilymi@hotmail.com) on 2016-03-22T01:17:39Z No. of bitstreams: 1 Mestrado-Emily Finall.pdf: 1593107 bytes, checksum: ce9ebf44b67e4020e069a84d708f2f71 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-22T17:56:40Z (GMT) No. of bitstreams: 1 bonfa_ec_me_sjrp.pdf: 1593107 bytes, checksum: ce9ebf44b67e4020e069a84d708f2f71 (MD5) / Made available in DSpace on 2016-03-22T17:56:40Z (GMT). No. of bitstreams: 1 bonfa_ec_me_sjrp.pdf: 1593107 bytes, checksum: ce9ebf44b67e4020e069a84d708f2f71 (MD5) Previous issue date: 2016-02-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As celulases podem ser utilizadas na bioconversão da fração de celulose de resíduos agro-industriais em açúcares fermentáveis, visando a obtenção de combustíveis renováveis e produtos químicos. As β-glicosidases são cruciais para a total sacarificação da celulose, mas na maioria dos casos elas são fortemente inibidas pelo seu produto, a glicose. Portanto, o conhecimento das cinéticas de hidrólise e as respostas dessa enzima frente a diferentes substratos e produtos pode definir a eficiência de hidrólise e do processo biotecnológico no qual poderia ser incorporada. O presente trabalho teve como objetivo caracterizar a β -glicosidase de 50 kDa (BG50) produzida pelo fungo termofílico Myceliophthora thermophila M.7.7 cultivado em estado sólido, em mistura de bagaço de cana e farelo de trigo (1:1). O zimograma do extrato bruto evidenciou duas isoformas, de aproximadamente 200 e 50 kDa, as quais foram separadas por cromatografia de filtração em gel. A caracterização da BG50 mostrou atividade ótima a 60 ˚C e pH 5,0 quando usado o 4-nitrofenol-β-D-glicopiranosídeo (pNPG), enquanto com celobiose o valor da temperatura e pH ótimo foram de 50 ˚C e pH 4,5, respectivamente. Testes realizados com adição de íons e reagentes mostraram diferenças nos efeitos sobre a atividade da enzima dependendo do substrato, principalmente com a adição de Ditiotreitol (DTT) utilizando celobiose, e inibição completa com Cu2+ e Fe3+ para pNPG e celobiose. Além disso, a enzima não mostrou efeito inibitório quando testada na presença de nove compostos fenólicos, uma característica significativa. Os estudos cinéticos revelaram um perfil de inibição competitiva pela glicose quando utilizado pNPG com valor de KI=1,5 mM e um Km significativamente menor (0,52 mM) pelo pNPG do que pela celobiose (Km=8,50 mM). Os parâmetros termodinâmicos mostraram que a BG50 é bastante estável, destacando seu tempo de meia vida de 855,6 minutos a 60 °C, porém desnatura facilmente acima dessa temperatura. Os resultados enfatizam a importância de investigar potencialidades de β-glicosidases baseadas na celobiose, uma vez que no processo industrial a enzima atuará sobre o substrato natural, além da compreensão da termoestabilidade da enzima. / Cellulases can be used in bioconversion of cellulose from agro-industrial waste into fermentable sugars in order to obtain renewable fuels and chemicals. The β-glucosidases are crucial to the overall saccharification of cellulose, but in most cases, they are strongly inhibited by its product, glucose. Therefore, knowledge of the hydrolysis kinetics of the enzyme and its responses against different substrates and products can set the hydrolysis efficiency and possible incorporation in biotechnological process. This study aimed to characterize the 50 kDa β-glucosidase (BG50) produced by the thermophilic fungus Myceliophthora thermophila M.7.7 grown in solid state, in a mixture of sugarcane bagasse and wheat bran (1:1). The zymogram of the crude extract showed two isoforms of 200 and 50 kDa, which were separated by gel filtration chromatography. The characterization of BG50 showed optimal activity at 60 °C and pH 5.0 when used pNPG, whereas using cellobiose the values of the optimal temperature and pH were 50 °C and pH 4.5, respectively. Tests with addition of reactants and ions showed differences in the effects on enzyme activity depending on the substrate, especially with the addition of dithiothreitol (DTT) utilizing cellobiose, and complete inhibition with Cu2+ and Fe3+ for 4-nitrophenyl-β- D-glucopyranoside (pNPG) and cellobiose. Furthermore, the enzyme showed no inhibitory effect when tested in the presence of nine phenolic compounds, a remarkable characteristic. Kinetic studies showed a profile of competitive inhibition by glucose when using pNPG with Ki = 1.5 mM and Km significantly lower (0.52 mM) with pNPG than using cellobiose (Km = 8.50 mM). The thermodynamic parameters show that BG50 is quite stable, highlighting its half life of 855.6 minutes at 60 ° C, but above this temperature easily denatured. The results emphasize the importance of investigating β-glucosidases’ potential based on cellobiose, since for the industrial processes the enzyme will function with its natural substrate, in addition to understanding the thermal stability of the enzyme.