Inositol dehydrogenase (IDH) catalyzes the oxidation of myo-inositol to scyllo-inosose using NAD+ as the coenzyme. IDH-related genes (Lp_iolG1 to Lp_iolG4) from Lactobacillus plantarum WCSF1 and (Lc_iolG1 and Lc_iolG2) from Lactobacillus casei BL23 were cloned into the vector pQE-80L, expressed in E. coli host cells and the proteins were purified to homogeneity. IDH activity of the purified enzymes was explored with myo-inositol and other structurally related compounds. It was found that IDH-related enzymes from L. plantarum WCSF1 did not exhibit any activity with tested substrates but, LcIDH1 and LcIDH2 from L. casei BL23 showed activity with myo-inositol and other related compounds. pH-rate profile studies have demonstrated the optimum pH for the reactions catalyzed by the active enzymes. Steady-state kinetics of the active enzymes was performed as with IDH from Bacillus subtilis (BsIDH), revealing that LcIDH1 is a myo-inositol dehydrogenase and LcIDH2 is a scyllo-inositol dehydrogenase. Both LcIDH1 and LcIDH2 are observed to be NAD+-dependent. Kinetic isotopic effect experiments for LcIDH1 have demonstrated that the chemical step in the reaction is partly rate-limiting. Substrate spectrum of LcIDH1 and LcIDH2 was explored and compared to BsIDH. Finally, a multiple sequence alignment of IDH-related enzymes was performed and the proposed consensus sequence motifs were considered to understand the activity differences between these enzymes.

ABSTRACT Protein-protein interactions play a central role in most biological processes. One such biological process is the maintenance of a reducing environment inside the cell. To maintain an internal reducing environment, living cells have evolved two enzymatic systems (glutathione and thioredoxin (Trx) systems). The Trx system is composed of the enzyme TrxR and its substrate Trx. The two proteins constitute an important thiol-dependent redox system that catalyzes the reduction of many proteins that are responsible for a variety of cellular functions. The system relies on transient protein-protein interactions between Trx and TrxR for its function. Cross-reactivity of components of the Trx system between species has been shown to be medically relevant. For example, Helicobacter pylori Trx (HP Trx) is thought to mediate catalytic reduction of human immunoglobulins and thus facilitate immune evasion. It has also been proposed that Helicobacter pylori gains access to the impenetrable gastric mucous layer by using secreted HP Trx to reduce the disulfide bonds present in the cysteine-rich mucin regions that are responsible for cross-linking mucin monomers. Therefore, disruption of secreted HP Trx-host protein interaction may result in restoration of the viscoelastic and hydrophobic protective properties of mucus. Previous studies aimed at understanding the nature of cross-reactivity of Trx system components among various species have shown that Trxs have higher affinity for cognate TrxRs (same species), than for TrxRs from different species. However, the basis for this specificity is not known. A growing body of evidence suggests that most protein-protein interactions are mediated by a small number of protein-protein interface residues, referred to as hot spot residues or binding epitopes. Therefore, understanding the biochemical basis of the affinity of proteins for their partners usually begins by identifying the hot spot residues responsible for the protein complex interactions. In this study, the crystal structures of Deinococcus radiodurans thioredoxin reductase (DR TrxR) and Helicobacter pylori TrxR (HP TrxR) were determined at 1.9 Å and 2.4 Å respectively. Analysis of the Trx-binding sites of both structures suggests that the basis of affinity and specificity of Trx for TrxR is primarily due to the shape rather than the charge of the surface. In addition, the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (EC Trx) was used to identify residues that are responsible for TrxR-Trx interface stability. Using computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface, 22 EC TrxR side chains were shown to make contact across the TrxR-Trx interface. Although more than 20 EC TrxR side chains make contact across the TrxR-Trx interface, our results suggest that only 4 residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent DR TrxR residues (M84, K137, F148, F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. These hot spot residues are surrounded by less important residues (hydrophobic and hydrophilic) that are also predicted to contribute to interface stability. F148 and F149 are invariant across bacterial TrxRs, however other residues that contact Trx are less conserved including M84 and K137. When M84 and K137 were changed to match equivalent E. coli TrxR residues (K137R, M84F); D. radiodurans TrxR substrate specificity was altered from its own Trx to that of E. coli Trx. The results suggest that a small subset of the TrxR-Trx interface residues are responsible for the majority of Trx binding affinity and specificity, a property that has been shown to general to protein-protein interfaces.

pH studies are used to gain insight into chemical mechanisms of enzyme catalysed reactions. However, perhaps the most important practical point that is often overlooked in pH studies is control of the ionic strength of reaction mixtures at the various pH values. For example, cathepsins Band L were suspected to be involved in cancer invasion but pH vs activity profiles indicated that they were not active at the extracellular pH (pH 7.2). When these profiles were re-evaluated in buffers of constant ionic strength, as opposed to buffers of constant molarity, it was shown that the enzymes were indeed active at pH 7.2. Other enzymes have also been reported to be sensitive to ionic strength. These include neutrophil elastase, class sigma glutathione S-transferase and penicillin G-acylase amongst others. The effects of increasing ionic strength on the activity of six enzymes were investigated. a-Glucosidase (from bakers ' yeast), elastase (human leukocyte) and trypsin (bovine pancreatic), cathepsin L (sheep liver), cathepsin B (rabbit liver), fruit bromelain (pineapple fruit) were subjected to different ionic strength buffers and their activities and Km and Vmax were determined as a function of ionic strength. The influence of ionic strength on Ki values has not been previously reported and was also studied, using the interaction between chicken egg-white cystatin C and cathepsin L as a model. a-Glucosidase was found to have an ionic strength optimum and elastase showed increasing activity with an increase in ionic strength. Trypsin activity decreased with increasing ionic strength with a substrate containing a positively charged side chain in the P1 position, and an increase in activity with a substrate containing a hydrophobic group at the P1 position. Cathepsin B activity increased when acting on the substrate Z-Phe-ArgNHMec and decreased when acting on Z-Arg-Arg-NHMec, with increasing ionic strength. Bromelain showed an increase in activity with increasing ionic strength. Cathepsin L activity decreased at increasing ionic strength and the Ki values for the cathepsin L-cystatin C interaction increased with increasing ionic strength. The results obtained can be attributed to the nature of the specificity pockets involved in binding the substrate, effects on the catalytic mechanism of the enzyme or structural changes due to increasing ionic strength. These results show that the ionic strength is a significant variable and should be kept constant or at in vivo levels when assaying enzymes. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Nitroreductase from Enterobacter cloacae (NR) is a member of a large family of homologues represented in all branches of the tree of life. However the physiological roles of many of these enzymes remain unknown. NR has distinguished itself on the basis the diverse sizes and chemical types of substrates it is able to reduce (Koder et al 1998). This might be an evolved characteristic suiting NR for a role in metabolism of diverse occasional toxins. While there are numerous studies of determinants of substrate specificity, we know less about mechanisms by which enzymes can be inclusive. Therefore, we present a synthesis of NR's dynamics, stability, ligand binding repertoire and kinetic mechanism. We find that NR reduces para-nitrobenzoic acid (p-NBA) via a simple mechanism limited by the chemical step in which the nitro group is reduced (Pitsawong et al 2014). Thus, for this substrate, NR's mechanism dispenses with gating steps that in other enzymes can enforce substrate specificity. Our data demonstrate that substrate reduction is accomplished by rate-contributing hydride transfer from the flavin cofactor coupled to proton transfer from solvent, but do not identify specific amino acids with a role. This is consistent with our crystal structures, which reveal a spacious solvent-exposed active site bounded by a helix that moves to accommodate binding of substrate analogs (Haynes et al 2002). Because it is able to reduce TNT (trinitrotoluene), herbicides and pesticides, NR has important potential utility in bioremediation.

Aromatic compounds comprise approximately one quarter of the Earth's biomass and thus play a critical role in the biogeochemical carbon cycle. These compounds are degraded almost exclusively by specialized microbial enzymes that are part of complex metabolic pathways. Detailed characterization of these enzymes is both a gateway to understanding a biological process fundamental to nature and a platform for bioengineering applications in bioremediation. Recently, a novel pathway was shown to metabolize two key aromatic intermediates: Benzoate and Benzoyl-Coenzyme A. Designated as the box pathway (benzoate oxidation), this metabolic conduit incorporates in succession; CoA-ligation, oxygenation, ring cleavage and neutralization of the aldehydic ring cleavage product, catalyzed by a Benzoate Coenzyme A Ligase (BCL), BoxAB, BoxC and an Aldehyde Dehydrogenase (ALDH) respectively. Collectively, these steps define the initial and unique segment of the box pathway. The objective of the research described here was to establish a molecular blueprint of the substrate binding pocket of the initial BCL and elucidate mechanistic details for both BoxC and ALDH enzymes from Burkholderia xenovorans LB400 through in-depth structural and functional characterizations. An intriguing feature of the box pathway in LB400 is a paralogous genetic organization. Functional studies on the BCL paralogs (BCLM and BCLC) show that BCLM is more active towards benzoate than BCLC. Structural analysis of the 1.84 Å resolution co- crystal structure of BCLM with benzoate reveals that the substrate binding pocket is closely contoured to bind benzoate, leaving little room to accommodate substituted benzoates, especially in the para position owing to a histidine (H339) residue that renders the pocket particularly shallow. Overall, while corroborative, the structural data provides a molecular rationale to our functional data where both the BCLs were seen to be highly specific for benzoate. Structural analysis of the 1.5 Å resolution crystal structure of the novel ring cleaving BoxC reveals an intriguing structural demarcation consistent with the primary sequence based divergence of BoxC within the crotonase superfamily. A highly divergent region in the C-terminus likely serves as a structural scaffold for the conserved N-terminus that harbors the active site. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site resulting in a compelling mechanistic model involving a pair of conserved glutamates (E146 and E168) and a novel cysteine (C111). Lastly, the 1.6 Å resolution co-crystal structure of ALDHC with NADPH and PEG allows identification of residues that are involved in rendering ALDHC selective for NADP+ and linear, medium to long chain aldehydes, as observed in our initial kinetic analyses. Functional and structural characterization of strategic ALDHC mutants enables us to propose a detailed reaction mechanism which involves the essential roles for C296 as the nucleophile, E257 as the general base and a proton relay network anchored by E496 and supported by E167 and K168. Overall, this research provides a molecular blueprint for three key box enzymes, thereby enhancing our understanding of central aromatic metabolism. / Graduate

Some yeasts secrete polypeptide toxins, which are lethal to other sensitive yeast cells, gram-positive pathogenic bacteria and pathogenic fungi. Therefore these are designated as killer toxins. Killer toxins are suggested as potent antimicrobial agents especially for the protection of fermentation process against contaminating yeasts, biological control of undesirable yeasts in the preservation of foods. Moreover they are promising antimicrobial agents in the medical field / due to immune system suppressing diseases like AIDS, there is an increase in the incidence of fungal diseases and current antimycotics have low selectivity and severe side effects. In this study our aim was to explain the cytocidal effect and enzymatic properties of K5 type yeast killer protein, which is secreted by Pichia anomala NCYC 434 cells, and known to have a broad range of killing spectrum. Competitive inhibition of the toxin with cell wall polysaccharides showed that primary binding site of toxin is &amp / #946 / -1,3-glucans of sensitive cells. Toxin showed exo-&amp / #946 / -1,3-glucanase activity which causes loss of cell wall rigidity leading cell death. Km and Vmax were found to be 0,3 mg/ml and 372,3 &micro / mol/min/mg for laminarin hydrolysis. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Toxin production was found to be dependent on &amp / #946 / -1,3-glucan content of the media. Toxin activity was completely inhibited by Hg+2 ,while several metal ions and DTT increased the activity to different extends. Our findings revealed the characteristics of K5 type killer toxin which will help for its possible uses in near future.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains xiv, 180 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

O intenso uso de compostos orgânicos em conjunto com o grande avanço industrial culminou em um enorme acúmulo de poluentes orgânicos no meio ambiente. Dentre estes poluentes têm-se destacado a presença de hidrocarbonetos aromáticos altamente tóxicos e resistentes à degradação física, química, fotolítica e biológica. Desta maneira, uma nova forma de combater a presença deste tipo de composto no meio ambiente têm sido estudada: o uso de microorganismos, naturais ou geneticamente modificados, capazes de transformá-los em substâncias inertes, como CO2 e água. Tal metodologia é denominada biorremediação. Dentres estes microorganismos destacam-se bactérias dos gêneros Pseudomonas, Aeromonas, Beijerinckia, dentre outros, que têm sido estudadas para esta finalidade. A enzima clorocatecol 1,2-dioxigenase (Pp 1,2-CCD) é uma das proteínas expressas por bactérias do gênero Pseudomonas putida, sendo responsável pela clivagem de hidrocarbonetos aromáticos através da incorporação de ambos os átomos de uma molécula de oxigênio à estrutura do anel aromático, sendo a proteína escolhida para desenvolvermos o presente trabalho. Mais especificamente, nos interessa estudar como o mecanismo de ação da referida enzima é controlado por moléculas extrínsecas, como fosfolipídios. Tal interesse pela interação entre a enzima e fosfolipídios surgiu recentemente quando da obtenção da primeira estrutura cristalográfica de uma enzima da família da CCD (dioxigenases intradióis). Nesta estrutura foi observado um sítio de ligação por monômero para fosfolipídios, o que fez com que várias questões relativas à influência desses sobre a atividade da enzima fossem levantadas. Nosso objetivo foi fazer uso das técnicas de Dicroísmo Circular (CD), Calorimetria e Ressonância Paramagnética Eletrônica (RPE) para estudar alterações conformacionais da enzima e de sua cinética induzidas por moléculas de fosfolipídio, e assim, obter informações que correlacionem as mudanças estruturais com o mecanismo de atividade enzimática da enzima. Os resultados obtidos através do uso daquelas técnicas em conjunto com protocolos que possibilitam a delipidação da enzima mostraram que a presença do fosfolipídios na estrutura da enzima tem influência sobre a atividade enzimática. Quando retiramos o fosfolipídio/ácido graxo, pudemos visualizar uma pequena mudança na estrutura secundária da enzima, um aumento da entalpia de reação, bem como um aumento na velocidade de reação, enquanto que a afinidade da enzima pelo substrato diminuiu. Pudemos também observar uma maior estabilidade térmica da enzima quando na ausência do fosfolipídio/ácido graxo e não foi observado interação da Pp 1,2-CCD com modelos micelares constituídos por lisofosfolipídios. Um breve estudo realizado sobre o papel da força iônica na atividade e na estabilidade térmica da proteína mostrou que na ausência de NaCl, em pH 8, a enzima se mostrou mais ativa, com uma afinidade pelo substrato maior e neste ambiente com baixa força iônica foi observado uma pequena interação da enzima com modelos micelares carregados negativamente. Assim, pudemos concluir que as moléculas anfipáticas, retiradas com os processos de delipidação, apesar de modificarem muito pouco a estrutura secundária da enzima, ainda assim instauram modificações na sua função de catálise do substrato catecol. Esta informação juntamente com os dados sobre inibição do processo reacional ocasionada pelo produto da reação formam um novo conjunto de dados que pode ser utilizado para se alcançar o objetivo mais geral de se controlar a atividade biológica da Pp 1,2-CCD. / The intensive use of organic compounds in conjunction with the industrial advances led to a huge accumulation of organic pollutants in the environment. Among these pollutants it has been noticed the presence of aromatic hydrocarbons that are highly toxic and resistant to physical, chemical and biological degradation. Thus, a new way to deal with the presence of this compounds in the environment has been studied: the use of microorganisms, natural or genetically modified, that can turn them into inert substances such as CO2 and water. This methodology is called bioremediation. Among those microorganisms, bacteria from the gender Pseudomonas, Aeromonas, Beijerinckia, among others, have been studied for this purpose. The enzyme chlorocatechol 1,2-dioxygenase (Pp 1,2-CCD) is one of the proteins expressed by Pseudomonas putida bacteria, being responsible for the cleavage of aromatic hydrocarbons through the incorporation of both atoms of a molecule of oxygen into the aromatic ring structure, being the protein chosen for investigation in this work. More specifically, we are interested in studying how the mechanism of action of this enzyme is controlled by extrinsic molecules such as phospholipids. The interest in the interaction between the enzyme and phospholipids arose recently when the first crystal structure of an enzyme of the intradiol dioxygenase family was reported. In this structure it was observed a binding site for a phospholipid per monomer, which raised many issues concerning its influence on the activity of the enzyme. Our goal was to use the techniques of Circular Dichroism (CD), calorimetry and Electron Magnetic Resonance (EMR) to study enzyme conformational changes and kinetics alterations induced by phospholipid molecules, thus gathering information on the structure-function correlation. The results obtained through those experimental techniques in conjunction with the use of protocols for protein delipidation showed that the presence of phospholipids/fatty acids in the structure of the enzyme play a role in enzyme activity. Upon removal of the phospholipid/fatty acids, we observed small changes in the secondary structure of the enzyme, an increase of the enthalpy of reactions as well as an increase in the reaction rate, whereas the affinity of the enzyme for the substrate decreased. We also observed a higher thermal stability of the Pp 1,2-CCD in the absence of the phospholipids/fatty acids, but no interaction was observed between the Pp 1,2-CCD and lysophospholipid micelles. A brief study of the function of ionic strength on the activity and thermal stability of the protein showed that in the absence of NaCl, at pH 8, the enzyme is more active, showing a greater affinity for the substrate and a low interaction was observed between Pp 1,2-CCD and negatively charged micelles. This information along with the data on the inhibition capacity of the reaction product are a new set of data that can be used to achieve the more general goal of controlling Pp 1,2-CCD biological activity.

IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...

The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...