Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity. The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C. The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible. Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c7dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS). Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions. A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU. Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c7dATP, dATPαS. / <p>QCR 20161027</p>

La levure Saccharomyces cerevisiae est capable d’utiliser une grande variété de sucres comme source de carbone et d’énergie. La plupart des enzymes impliquées dans l’utilisation de ces sucres sont codées par des gènes issus de familles multigéniques. C’est le cas de la famille IMA identifiée comme impliquée dans l’utilisation de l’isomaltose. Cette famille comprend cinq gènes qui codent pour quatre isomaltases partageant une forte identité de séquence (de 65% à 100 %). Dans ce travail , la diversitéfonctionnelle de la famille IMA a été étudiée, en caractérisant de façon exhaustive in vitro leurs propriétés biochimiques et enzymologiques. Ima1p et Ima2p possèdent des propriétés biochimiques identiques (pH, température, et thermostabilité) mais Ima3p se distingue par rapport à ces deux protéines bien que n’ayant que trois acides de différence avec Ima2p (thermostabilité plus faible). Ima5p quant à elle, est la protéine la plus dissemblable (température optimale plus faible et demi-vie basse dès 37°C). Les quatre isomaltases sont cependant très sensibles au Tris et aux ions Fe3+. Les quatre isoenzymes présentent une préférence pour les disaccharides liés en α-1,6 (isomaltose et palatinose), avec une cinétique de type Michaëlis-Menten et une inhibition par le substrat à une concentration élevée. Les isomaltases Imap sont cependant aussi capables d'hydrolyser les disaccharides α-1,2, α-1,3 et α-1,5 ainsi que les trisaccharides portant une liaison α-1,6, ce qui met en évidence leur ambiguïté de substrat .Nos résultats ont toutefois montré de nombreuses singularités dans cette famille de protéines. Alors que Ima1p et Ima2p présentent des propriétés très semblables, l’activité catalytique de Ima3p est globalement très faible malgré sa forte ressemblance avec Ima2p. Le variant Ima3p_R279Q retrouve des niveaux d'activité proches de ceux d’Ima2p, tandis que la substitution d’une leucine par une proline à la position 240 a permis d’augmenter de manière significative la stabilité d’Ima3p confirmant le rôle des prolines dans la thermostabilité des protéines. L’hydrolyse de l’isomaltose par Ima5p réfute lesconclusions précédemment publiées sur l'exigence d'acides aminés spécifiques pour déterminer la spécificité de α-1,6 puisque le variant IMA5-MQH ne permet pas de restaurer une activité semblable à Ima1p malgré la présence des trois résidus MQH. Nous avons également trouvé qu’Ima5p est inhibé par le maltose suivant une inhibition mixte tandis qu’Ima1p est inhibée de façon compétitive à faible concentration et de manière incompétitive à forte concentration en isomaltose / Most enzymatic systems for sugar uptake and assimilation rely on multigene families in theyeast Saccharomyces cerevisiae. The IMA / MAL family has been used as a model system to study themolecular mechanisms that govern evolution of duplicated genes. The five IMA multigene familymembers encode four isomaltases sharing high sequence identity from 65% to 99%, of which IMA3and IMA4 are 100% identical to encode the same isomaltase. In this work, the functional diversity ofIMA family was further explored, with exhaustive in-vitro characterization of their biochemical andenzymological properties.Ima1p and Ima2p were similar to biochemical properties; Ima3p showed some differences fromthe two proteins; amongst them, Ima5p was the most distant protein. The four isomaltases were highlysensitive to Tris and Fe3+, but were unaffected by the addition or the removal of Ca2+ despiteconservation of the calcium binding site. Besides, four isoenzymes exhibited a preference for the α-(1,6)disaccharides isomaltose and palatinose, with Michaelis-Menten kinetics and inhibition at highsubstrates concentration. They were also able to hydrolyse trisaccharides bearing an α-(1,6) linkage,but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrateambiguity. While Ima1p and Ima2p presented almost identical characteristics, the results neverthelessshowed many singularities within this protein family. In particular, Ima3p presented lower activitiesthan Ima2p despite only 3 different amino acids between these two isoforms. The Ima3p_R279Qvariant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240significantly increased the stability of Ima3p and supported the role of prolines inthermostability.Ima5p presented the lower optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specificamino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibitionby maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitiveat very low isomaltose concentrations and uncompetitive as the substrate concentration increased.The presented Ph.D’s work provided preliminary insights into determining structural factorswithin this family, exemplifying for example the role of proline residues for thermosability. Moreover,it was illustrated that a gene family encoding proteins with strong sequence similarities can lead toenzyme with notable differences in biochemical and enzymological properties.

A fim de garantir a viabilidade econômica da produção de etanol de segunda geração é necessário o desenvolvimento de tecnologias eficientes para a hidrólise enzimática dos materiais lignocelulósicos. Além disso, o elevado consumo de água pelas biorrefinarias tem despertado grande atenção para a utilização de recursos hídricos não-potáveis, como a água do mar. Assim, atualmente busca-se por enzimas tolerantes a altas concentrações salinas, bem como aos subprodutos gerados e/ou acumulados nas etapas de pré-tratamento da biomassa. Nesse contexto, o objetivo deste trabalho foi a purificação e caracterização cinética e bioquímica de uma ß-xilosidase produzida por uma linhagem do fungo mesófilo Colletotrichum graminicola. A enzima purificada (Bxcg) apresentou conteúdo de carboidratos totais de 54% (m/m), ponto isoelétrico de 4,2 e uma massa molecular aparente de cerca de 130 kDa, que foi reduzida para cerca de 92 kDa após deglicosilação. A enzima mostrou boa tolerância a elevadas concentrações de sal e manteve cerca de 90% da atividade controle na presença de NaCl 0,5 mol L-1 (concentração média de NaCl na água do mar). A temperatura e pH ótimos de reação foram 65 ºC e 4,5, respectivamente, tanto na ausência quanto na presença de NaCl 0,5 mol L-1. Já na presença de NaCl 2,5 mol L-1 o pH ótimo de atividade foi alterado para 5,0. Bxcg permaneceu estável numa ampla faixa pH (4,0 - 7,5) tanto na ausência quanto na presença de sal. A enzima mostrou ótima estabilidade térmica e manteve completamente estável à 50 ºC após 24 horas de incubação. A presença de elevada concentração de NaCl (2,5 mol L-1) resultou num aumento na termoestabilidade da enzima. A atividade enzimática foi tolerante aos íons Ca2+, Sr2+, Co2+, Zn2+, Ni2+, Mn 2+, Mg2+, K+ e Na+. Na ausência de sal, Bxcg hidrolisou p-nitrofenil-?-D-xilopiranosídeo (pNP-XIL) com Vmáx de 348,8 ± 11,5 U mg-1, KM de 0,52 ± 0,02 mmol L-1 e alta eficiência catalítica (kcat/KM = 1432,7 ± 47,3 L mmol-1 s-1). Em presença de sal, a afinidade aparente de Bxcg pelo substrato foi levemente menor e a hidrólise ocorreu com Vmáx menor, resultando em eficiência catalítica cerca de 1,5 de vezes menor, se comparadas as condição de ausência de sal. A enzima apresentou atividade bifuncional de ?-xilosidase/?-L-arabinofuranosidase. Bxcg hidrolisou p-nitrofenil-?-L-arabinopiranosídeo com afinidade aparente cerca de 18 vezes menor (KM = 9,6 ± 0,5 mmol L-1) que a estimada para pNP-XIL e a hidrólise do substrato ocorreu com Vmáx de 148,4 ± 4,4 U mg-1 e eficiência catalítica de 33,1 ± 1,6 L mmol-1 s-1. A enzima foi fortemente inibida por xilose com KI de 3,3 mmol L-1. Bxcg foi capaz de hidrolisar xilooligossacarídeos até xilohexaose, inclusive aqueles com ramificação de ácido 4-O-metilglucurônico. Bxcg e uma endo-xilanase purificada do mesmo microrganismo apresentaram um forte efeito sinérgico (3,1 vezes) para hidrólise de xilana beechwood. A enzima mostrou-se tolerante aos solventes butanol, glicerol, tolueno e acetona, bem como aos surfactantes Triton X-100, Tween 80 e Tween 20, enquanto que o líquido iônico acetato de 1-etil-3-metilimidazólio inibiu fortemente a atividade enzimática. De uma maneira geral, Bxcg apresenta propriedades atraentes para a aplicação em processos de sacarificação da biomassa lignocelulósica, incluindo aqueles conduzidos em elevada salinidade e/ou em presença de compostos residuais gerados ou acumulados nas etapas de pré-tratamento da biomassa / In order to ensure the economic viability of the production of second-generation ethanol, it is necessary the development of efficient technologies for the enzymatic hydrolysis of lignocellulosic materials. In addition, the large consumption of water by biorefineries has attracted great attention for the use of non-potable water resources, such as seawater. Therefore, enzymes tolerant to high salt concentrations and the by-products generated and/or accumulated in the biomass pretreatment steps are widely studied. In this context, the objective of this study was the purification and kinetic and biochemical characterization of a ?-xylosidase produced by a strain of the mesophilic fungus Colletotrichum graminicola. The pure enzyme (Bxcg) showed a total carbohydrate content of 54% (w/w), isoelectric point of 4.2 and an apparent molecular weight of 130 kDa, which was reduced to 92 kDa after deglucosylation. The enzyme showed good tolerance to high salt concentrations and retained aproximately 90% of the control activity in the presence of 0.5 mol L-1 NaCl (NaCl concentration in seawater). The optimum reaction temperature and pH were 65 °C and 4.5, respectively, both in the absence and presence of 0.5 mol L-1 NaCl. In the presence of 2.5 mol L-1 NaCl, the optimum pH was altered to 5.0. Bxcg retained stable over a wide pH range (4.0 - 7.5) both in the absence and presence of salt. The enzyme showed excellent thermal stability and retained completely stable at 50 °C after 24 hours of incubation. The presence of high NaCl concentration (2.5 mol L-1) resulted in an increase in the thermostability of the enzyme. The enzymatic activity was tolerant to Ca2+, Sr2+, Co2+, Zn2+, Ni2+, Mn2+, Mg2+, K+ and Na+. In the absence of salt, Bxcg hydrolyzed p-nitrophenyl-?-D-xylopyranoside (pNP-XIL) with Vmax of 348.8 ± 11.5 U mg-1, KM of 0.52 ± 0.02 mmol L-1 and high catalytic efficiency (kcat/KM = 1432.7 ± 47.3 L mmol-1 s-1). In the presence of salt, the apparent affinity for the substrate was slightly lower and the hydrolysis occurred with smaller Vmax, resulting in catalytic efficiency 1.5 fold lower, when compared to the salt. The enzyme showed bifunctional ?-xylosidase/?-L-arabinofuranosidase activity. Bxcg hydrolyzed p-nitrophenyl-?-L-arabinopyranoside with apparent affinity 18-fold lower (KM = 9.6 ± 0.5 mmol L-1) than that estimated for pNP-XIL and substrate hydrolysis occurred with Vmax of 148.4 ± 4.4 U mg-1 and catalytic efficiency of 33.1 ± 1.6 L mmol-1 s-1. The enzyme was strongly inhibited by xylose with KI of 3.3 mmol L-1. Bxcg was able to hydrolyze xylooligosaccharides from xylohexaose, including those with 4-O-methyl-glucuronic acid branch. Bxcg and a pure endo-xylanase from the same microorganism had a strong synergistic effect (3.1 fold) for hydrolysis of xylan beechwood. The enzyme was tolerant to the butanol, glycerol, toluene and acetone solvents, as well as the Triton X-100, Tween 80 and Tween 20 surfactants, whereas the 1-ethyl-3-methylimidazolium acetate ionic liquid strongly inhibited the enzymatic activity. In summary, Bxcg has attractive properties for application in saccharification processes of the lignocellulosic biomass, particularly under high salinity and/or in the presence of residues of biomass pretreatment steps

Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal β-, bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable roperties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While he factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood. The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX. This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms. In addition, the candidate has carried out the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain Cα and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins. Chapter 1 starts with a brief introduction of xylanases, their classifications and overall folds. At present, a little more than a dozen of crystal structures of GH10 xylanases are known and described in the literature. A brief mention about these structures and their optimum pH and temperature is outlined under a separate section. In view of the industrial importance of the study enzyme, the potential industrial and biotechnological applications of xylanases are detailed in this Chapter. A section is dedicated to describe the present study enzyme BSX, an alkali thermostable endo-xylanase from an alkalophilic bacterium, Bacillus sp. NG-27. BSX has a molecular mass of ~41 kDa and is optimally active at 343 K and at a pH of 8.4. The alkaline thermostability of the wild type BSX is likely to be industrially important. At the end, the scope of the present work is detailed. Chapter 2 presents the purification of xylanase (BSX) from Bacillus sp. NG-27, the crystallization of the native and xylosaccharides-bound BSX, the X-ray diffraction data collection on these crystals and processing of the data. Repeated attempts to crystallize the protein expressed in the chloroplast of transgenic tobacco plant were unsuccessful. However, crystallization was achieved with the protein sample purified from the native source by hanging drop vapour diffusion method. Crystals were grown at both acidic (4.6) and basic pH (8.5). The corresponding crystallization conditions are 0.2 M MgCl2, 0.1 M sodium acetate pH 4.6 and 20% PEG 550 MME and 0.1 M aCl, 0.01 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000. Crystals grown at acidic pH were not suitable for X-ray diffraction study. Subsequently, crystal obtained at a basic pH of 8.5 was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). Native BSX crystals belong to monoclinic space group C2 with unit cell parameters a = 174.5 Å, b = 54.7 Å, c = 131.5 Å and β = 131.2°. Crystals of xylosaccharides-bound enzyme were grown in a slightly modified crystallization condition of native, 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000 and the enzyme was incubated with xylan prior to setting up the crystallization. Crystals belong to primitive orthorhombic space group P212121 with unit cell parameters a = 59.2 Å, b = 83.8 Å and c = 174.4 Å. A data set was collected using synchrotron radiation of wave length 1.0 Å from a cryo-cooled crystal at Spring-8 BL26B1 beam line, Japan. The Matthews coefficient VM for native and xylosaccharides- bound crystals was calculated to be 2.8 and 2.7 Å3 Da-1, respectively, suggesting two molecules in each crystal asymmetric unit. No twinning was detected in both the datasets and the overall quality of the data sets was found to be good. Chapter 3 details the application of molecular replacement method to the structure solution of native and xylosaccharides-bound BSX, the course of iterative model building and the refinement carried out, and the quality of the final protein structure models. The native-enzyme structure solution was obtained by the molecular replacement method using as a search model the crystal structure (PDB code 1hiz) of the closest homologous, extracellular xylanase (GSX) from Geobacillus stearothermophilus. No non- crystallographic symmetry (NCS) restraint was applied between the two independent molecules in the crystal asymmetric unit at the final round of refinement. The final positional refinement of native BSX converged to R factors of R = 19.4% and Rfree = 23.5% for data between 20.0 to 2.2 Å. The final native model consists of 5704 protein atoms, two Mg2+ ions and 721 solvent water molecules. The final native model was taken as the search structure for the xylosaccharides-bound BSX and a solution with a correlation coefficient of 70.7% and an R-factor of 32.1% was obtained from the molecular replacement calculation. Unlike the native structure refinement, NCS restraint was imposed at all stages of the refinement. Bound xylosaccharides were clearly visible inthe difference Fourier electron density maps. The last round of refinement gave a model with R and Rfree of 21.8% and 25.7%, respectively. The final xylosaccharides-bound model consists of 5766 protein atoms, four Mg2+ ions, 85 atoms belong to bound xylosaccharides and 523 solvent water molecules. No residues were found in the disallowed region of the Ramachandran (φ, ψ) map for both the structures. Chapter 4 describes the native and xylosaccharides-bound BSX crystal structures and the structural comparison of BSX with other GH10 family xylanase crystal structures for which the optimum temperature and pH are known in the literature. BSX folds as the ubiquitous (β/α)8-barrel, a common structural superfold characteristic of GH10 xylanases. The two active site glutamic acid residues, Glu149 and Glu259, are located on opposite sides of the active site cleft and their side-chains are at a distance of 5.5 Å apart suggesting the enzymatic reaction takes place by the retaining mechanism. From the structural superposition of other xylotriose-bound xylanase structures on to the xylosaccharides-bound BSX, structural plasticity in the xylotriose binding can be inferred, implying that the xylose recognition at the subsite -3 displays plasticity and is less specific as opposed to that at -1 and -2 subsites. The stacking interaction of one of the xylose moieties of the xylobiose with the Trp235 seen in BSX provides, for the first time, a structural evidence for the direct involvement of Trp235 in xylosaccharides binding. The crystal structure revealed a metal binding site, found at the C-terminal end of catalytic domain. The presence of metal binding site was not anticipated from earlier theoretically modeled structure and biochemical studies. Further, we have shown experimentally the requirement of Mg2+ ion for the enzyme activity. We havedescribed a novel WP sequence-structure-interaction motif which is present in the (+) side of the active site region and presumably helps in the efficient binding of the carbohydrate moiety of the xylan in the active site cleft of BSX. The structural comparison of BSX with other GH10 xylanases solved to date and characterized to be active at a pH close to neutral was done for the first time. The comparative study revealed the essential structural features which may responsible for the alkaline stability of GH10 xylanases.Briefly, the alkalophilic GH10 xylanases from alkalophilic organisms have surface abundant in acidic residues, the heat and alkaline susceptible residue Asn depleted on the protein surface and increased number of salt bridges. Our study has unveiled the role of the nature and composition of protein surface amino acids in the adaptation of enzymes to polyextreme conditions. The observations reported in the thesis provide important lessons for engineering alkaline stability in xylanases for industrial applications and in general for the understanding of alkaline stability in related proteins. A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in polyextremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. Chapter 5 summaries the important findings of the present study from the crystal structural analysis of BSX and its comparison with non-alkalophilic GH10 xylanases. Separate sections are made on conclusions and future prospects for the study on BSX. Chapter 6 describes the comprehensive database analysis of C-H…O hydrogen bond in helices of globular proteins. The C-H…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1.Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 C- H…O hydrogen bonds involving methylene Cγ1 atom as donor in α-helices. In addition, C-H…O hydrogen bonds are present along with helix stabilizing salt bridges and to some extent compensate for the side-chain conformational entropy loss. Our analysis highlights that a multitude of local C-H…O hydrogen bondsformed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini. A majority of the helix favouring residues, Met, Glu, Arg, Lys, Leu and Gln which also have large side-chains with more donatable CH groups, have significant propensity to form side-chain to main-chain C-H…O hydrogen bonds in helix. The large side-chains are marked by their ability to shield from the solvent the polar atoms of the peptide backbone and at the same time participate in weak cohesive C-H…O interactions in the helix. This chapter also details the identification for the first time a novel chain reversal motif stabilized by 1 → 5 Cα-H…O interactions. The importance of these hydrogen bonds with respect to helix stability is discussed in the final section of the chapter. Appendix A details the crystallographic and structural analyses oftwares used for the present thesis work. Appendix B describes, in addition to the crystal structure analysis of BSX, the work carried out by the candidate on a comparative study of a thermostable xylanase from Thermoascus aurantiacus, solved in our laboratory at atomic 1.11 Å (293 K) and ultrahigh 0.89 Å (100 K) resolutions. From the comparison, we have for the first time pointed out the possibility of plasticity of ion pairs in proteins with water molecules mediating some of the alternate arrangements. The αβ-loops are relatively less flexible than the βα-loops. The β-strands are least affected structurally with the increase in temperature. Thus the TIM barrel fold in the study enzyme, though having a single domain, may be dissected into parts based on the relative flexibility and described as having a rigid core constituted by the β-barrel and a less rigid exterior formed by the surrounding α-helices. Appendix C presents the crystallization and the preliminary X-ray characterization work done by the author of the thesis on an alkali thermostable cellulase enzyme from Thermomonospora sp. The protein is an extracellular enzyme with molecular mass of 14.2 kDa and interestingly, has the dual activity for both cellulose and xylan. The primary structure of the enzyme is not known. The enzyme was purified from the source organism and crystallized. A complete diffraction data set was collected and processed to 2.3 Å in an orthorhombic space group P212121. Appendix D contains tables which give details about the analysed 5 → 1 Cα- H…O hydrogen bonds in helices and a novel chain reversal motif with 1 → 5 Cα-H…O hydrogen bonds. Appendix E encloses reprints of publications which have resulted from the work reported in the thesis.

<p>Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.</p><p>The wild-type North American firefly<i>(Photinus pyralis)</i>luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.</p><p>The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.</p><p>Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).</p><p>Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.</p><p>A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.</p><p><b>Key words:</b>bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.</p>

Les phytases hydrolysent les phytates représentant la forme majeure de stockage du P dans les céréales. Ces phytates sont aussi des facteurs anti-nutritionnels qui chélatent les cations réduisant leur absorption. Dans le premier volet de cette thèse, une nouvelle souche bactérienne produisant une phytase extracellulaire a été isolée et identifiée comme Bacillus amyloliquefaciens US573. L’enzyme «PHY US573» a été purifiée et caractérisée en comparaison avec deux phytases commerciales Ronozyme PL et Natuphos. PHY US573 se distingue par sa forte thermostabilité en présence de calcium. En outre, PHY US573 se caractérise aussi par une tolérance remarquable aux sels comme le NaCl et LiCl. L’ensemble de ces propriétés montre que PHY US573 pourrait être une candidate intéressante pour des applications en alimentation animale ou en agriculture pour améliorer la biodisponibilité du P-phytique pour les plantes. Dans le deuxième volet, la souche Streptomyces sp. US42 produisant une activité phytase extracellulaire a été sélectionnée. L’enzyme «PHY US42» a été purifiée et caractérisée. PHY US42 est calcium dépendante également une grande stabilité en présence de sels biliaires et des protéases digestives. La modélisation moléculaire de PHY US42 indique qu'elle appartient au groupe des β-propeller phytases qui sont généralement calcium-dépendantes. Vu ses propriétés biochimiques intéressantes, PHY US42 constitue une bonne candidate comme additif dans les aliments pour animaux monogastriques en combinaison avec une histidine acide phytase. Enfin dans un troisième volet, nous nous sommes intéressés à l’étude de la régulation de l'expression du gène phytase de S. coelicolor M145 (sco7697) chez S. coelicolor M145, S. lividans TK24 ainsi que chez ses deux mutants ppk et phoP. Ainsi, en plus des boites pho localisées en amont de la région promotrice -35 siège de la régulation positive PhoP-dépendante, nous avons révélé pour la première fois que la RD localisée en aval de la région promotrice -10 est le siège d’une forte régulation négative par un répresseur inconnu. Ce dernier empêcherait l’activation PhoP-dépendante de l’expression du gène phytase. / Phytases hydrolyse phytate representing the major storage form of P in cereal. phytates are also anti-nutritional factors that chelate cations such as Ca²⁺, Mg²⁺, Fe²⁺, Z²⁺ reducing their absorption. The low bioavailability of phytic phosphorus in monogastric animals require their food supplementation with Pi to meet the needs of the animal in P. This creates an extra cost and increases the environmental pollution by the manure excretion highly charged phosphate. In the first part of this thesis, from soil samples taken near hot hydrothermal waters of the region Elhamma in southern Tunisian, a new bacterial strain producing extracellular phytase was isolated and identified as Bacillus amyloliquefaciens US573. The enzyme referred "PHY US573" was purified and characterized in comparison with two commercial acid histidine phytases Ronozyme PL and Natuphos. PHY US573 is calcium dependent and has an optimum activity at pH 7.5 (5 for Ronozyme and 5.5 for Natuphos) and 70°C (55°C for Ronozyme and Natuphos). PHY US573 is distinguished by its high thermostability, in fact, it keeps 93% of its activity after incubation for 10 min at 75°C in the presence of calcium while Ronozyme and Natuphos keep only 45% and 53% of their activity, respectively. This enzyme is specific for phytic acid and also has a very good stability at pH 3 to 9 and a perfect stability in presence of bile salts. In addition, PHY US573 is also characterized by a remarkable salt tolerance because it retains 80 to 95% of its activity in the presence of 20 g/l of NaCl and LiCl, respectively. All these properties shows that PHY US573 could be an interesting candidate for applications in feed industry alone or in combination with an histidine acid phytase. In a second part of this thesis, from the Streptomyces collection of LMB-CBS, a strain producing extracellular phytase activity was selected and identified as Streptomyces sp. US42. The enzyme "PHY US42" was purified and characterized. PHY US42 has a calcium-dependent activity (such as Bacillus phytases), optimally active at pH 7 and 65°C. PHY US42 is perfectly stable at pHs ranging from 5 to 10 and its thermal stability is greatly increased in the presence of calcium. Indeed, PHY US42 maintains 80% of its activity after 10 min of incubation at 75 °C in the presence of calcium. PHY US42 has also a high stability in the presence of bile salts and digestive proteases. Molecular modeling of PHY US42 indicates that it belongs to the β-propeller phytase group which are usually calcium-dependent. Given its interesting biochemical properties, PHY US42 which would operate mainly in the intestine, is a good candidate for use as an additive in agastriques fish food or in combination with an histidine acid phytase in feed industry. Finally in a third part, we are interested in studying the regulation of the expression of the phytase gene of S. coelicolor M145 (sco7697) in S.coelicolor M145, S.lividans TK24 and among its two mutants ppk and phoP. To do this, we merged the wild promoter regions (phyWT) or mutated (phym1, phym2, phym1+2) of sco7697 gene with the GUS reporter gene encoding ß-glucuronidase activity. Thus as expected, we demonstrated that the deletion of the PHO box located upstream of the -35 reduces the level of induction of sco7697 in conditions of Pi limitation. Moreover, we have revealed for the first time that the alteration of RD located downstream of -10 correlates with a dramatic increase of GUS expression when PhoP is present. Our results demonstrate that this RD is the seat of a strong negative regulation by an unknown repressor. This would prevent the PhoP-dependent activation of expression of the phytase gene.

L’usage excessif d’antibiotiques a provoqué l’émergence de résistance, constituant un problème sanitaire mondial. L’antibiotique triméthoprime (TMP) inhibe l’enzyme dihydrofolate réductase (FolA) des bactéries, interrompant la production d’un précurseur essentiel dans la synthèse des purines et empêchant ainsi la croissance bactérienne. Cependant, certaines bactéries produisent une seconde dihydrofolate réductase : une DfrB, appartenant à une famille d’enzymes hautement résistantes au TMP. Actuellement, dix membres de la famille DfrB ont été identifiés, qui partagent une identité de séquence élevée (74 – 98 %). Les enzymes DfrB sont constituées de domaines identiques de 78 acides aminés, de type ‘SH3-like’, qui s’homotétramérisent afin de former l’enzyme active. Les DfrB ne partagent aucune homologie de séquence ou de structure avec les FolA et aucun antibiotique n’a encore été développé pour contourner la résistance au TMP causée par les DfrB. Afin de mieux comprendre le domaine SH3-like, des homologues (DfrB-H) partageant 10 à 80 % d’identité avec la DfrB1 ont été identifiés et caractérisés. Ils possèdent une activité dihydrofolate réductase (Dfr) et confèrent de la résistance au TMP. De plus, afin de vérifier si les gènes dfrB se retrouvent dans divers environnements, une recherche dans une base de données métagénomiques a été entreprise, permettant de caractériser 10 nouvelles séquences homologues aux DfrB connues. En 2012, le groupe Pelletier a rapporté le premier inhibiteur spécifique d’une DfrB, et plusieurs autres depuis. Seule la DfrB1 a été caractérisée concernant son profil d’inhibition ainsi que sa thermostabilité inhabituelle. Ici, une méthode semi-automatisée sera développée pour caractériser les profils d’inhibition, de thermostabilité, de résistance au TMP et d’activité enzymatique de toutes les DfrB et des homologues identifiés, afin de les comparer à ceux de la DfrB1. Pour atteindre ces objectifs, des nouvelles méthodes à haut débit de détermination d’activité ainsi que des tests de concentration minimale inhibitrice (CMI) furent développés. Ces méthodes ont permis de déterminer que les profils de thermostabilité et d’inhibition de plusieurs DfrB et DfrB-H sont comparables aux profils de la DfrB1. De plus, le criblage de dizaines de composés potentiellement inhibiteurs a été effectué afin de poursuivre la recherche d’inhibiteurs spécifiques aux DfrB. En outre, nous signalons 10 nouvelles séquences homologues de DfrB qui confèrent une résistance élevée au TMP et possèdent une activité Dfr. La caractérisation de tous les membres DfrB et les homologues nous permettra d’acquérir une meilleure connaissance de leur mécanisme de résistance, de leur prévalence dans divers environnements et de soutenir notre développement de nouveaux inhibiteurs des DfrB. / The intensive usage of antibiotics has provoked the emergence of antibiotic resistance, causing a worldwide health issue. The antibiotic trimethoprim (TMP) targets the microbial dihydrofolate reductase enzyme (FolA), abrogating the production of an essential precursor in the synthesis of purines and thus preventing bacterial proliferation. However, some bacteria produce an additional dihydrofolate reductase: the highly TMP-resistant DfrB. Currently, ten DfrB family members have been identified, that share high sequence identity (74 – 98 %). DfrB enzymes consist of identical, 78 amino acid-long SH3-like domains, that homotetramerize to form the active enzyme. DfrB share no sequence or structural homology with FolA and no antibiotic has yet been developed to circumvent the TMP resistance caused by DfrB. In order to gain insight into the SH3-like domain of DfrB, homologues (DfrB-H) sharing 10 to 80 % identity with DfrB1 were identified and characterized, which displayed dihydrofolate reductase (Dfr) activity and conferred high TMP resistance. Also, to investigate if dfrB genes are identified in various environments, a metagenomic database search was undertaken to characterize ten new DfrB1 homologue sequences. In 2012, the Pelletier group reported the first specific inhibitor of a DfrB, and several others since. Only DfrB1 has been characterized regarding its inhibition profile as well as its unusual thermostability. Here, semi-automated methods will be developed to compare the inhibition, thermostability, TMP-resistance and enzymatic activity profiles of all DfrB and DfrB homologues to those of DfrB1. To address this objective, new high-throughput activity assays as well as Minimal Inhibitory Concentration (MIC) assays were developed. Using those methods, we determined that thermostability and inhibition profiles of several DfrB and DfrB-H were comparable to those of DfrB1. Also, a screen of several dozen potential inhibitory compounds was performed, to attempt to identify further specific DfrB inhibitors. In addition, we report 10 new DfrB homologues that confer high TMP resistance and possess Dfr activity. The characterization of all DfrB members and DfrB homologues will allow us to acquire greater knowledge on their antimicrobial resistance mechanism, their prevalence in different environments and support our development of new DfrB-specific inhibitors.

Användningen av biokatalysatorer, särskilt enzymer, inom kemikalie- och läkemedelsindustrin erbjuder betydande fördelar jämfört med de traditionella kemo-katalytiska metoderna som historiskt har dominerat industrin. En viktig klass av enzymer, transaminaser, spelar en central roll i tillverkningen av kirala aminer, som utgör grundläggande byggstenar i dessa industriella sektorer. Denna studie är inriktad på ett specifikt amin transaminas från Silicibacter pomeroyi. Tidigare har detta enzym visat förmåga att katalysera en mängd olika reaktioner för produktion av kirala aminer, men för att realisera dess fulla potential inom industriella tillämpningar krävs förbättrad stabilitet vid högre temperaturer. I motsats till de vanligt förekommande metoderna för proteinteknik, såsom rationell design och riktad evolution, används i denna studie släktsekvensrekonstruktion för att skapa mer temperaturtåliga varianter av detta enzym. Tidigare användning av denna metod har visat sig kunna generera proteiner med högre temperaturtålighet. Genom denna metod, där förfäder till detta enzym återskapas utifrån bevarade sekvenser, förväntas generering av varianter som kan bibehålla sin funktion vid högre temperaturer under en längre tid. Genom att utforska denna alternativa strategi för proteinteknik strävar studien efter att ge mer robusta biokatalysatorer för industriella tillämpningar. Utfallet från denna studie visade att två förfäder hade ökad termostabilitet. Detta visade sig dels genom analys av T5015 som påvisade en 3.9 och 6 C° förbättring för respektive förfader. Vidare påvisade t1/2 mätningar att dessa enzymer kunde utstå 2.06 till 3.72 gånger så lång tid vid 55 C° innan de inaktiverades. De påvisade dock lägre specifik aktivitet vid rumstemperatur, där en bidragande faktor till detta var att enbart en liten fraktion av förfäderna är korrekt veckade. Detta visar att det är nödvändigt med fortsatta förbättringar och fortsatta studier kring substratacceptans och stabilitet i olika lösningsmedel. Sammanfattningsvis påvisar resultaten att släktsekvensrekonstruktion är en proteinteknik som fungerar för att skapa proteiner med ökad termostabilitet och bör ses som ett mer självklart alternativ till riktad evolution och rationell design. / The utilisation of biocatalysts, particularly enzymes, in chemical and pharmaceutical industries presents significant advantages over the traditional chemocatalytic methods that historically dominated the industry. A crucial class of enzymes, transaminases, play a central role in the production of chiral amines, fundamental building blocks in these industrial sectors. This study focuses on a specific amine transaminase from Silicibacter pomeroyi. While this enzyme has previously demonstrated the ability to catalyse a variety of reactions for chiral amine production, realising its full potential in industrial applications requires enhanced stability at higher temperatures. In contrast to commonly employed protein engineering methods such as rational design and directed evolution, this study utilises ancestral sequence reconstruction to generate more temperature-resistant variants of this enzyme. Previous applications of this method have shown promising results in generating proteins with increased thermal stability. Through this approach, wherein ancestors of this enzyme are recreated from extant sequences, it is expected that variants capable of maintaining function at higher temperatures will be produced. By exploring this alternative strategy for protein engineering, the study aims to provide more robust biocatalysts for industrial applications. The outcome of this study is that two ancestors exhibited increased thermostability. This was evidenced by the analysis of T5015, which showed an improvement of 3.9 and 6 °C for each respective ancestor. Furthermore, t1/2 measurements indicated that they remained active for 2.06 to 3.72 fold longer at 55 °C before becoming inactive. However, they exhibited lower specific activity at room temperature, partially due to only a small fraction of the ancestral protein samples being properly folded. This suggests further improvements and continued investigations into substrate acceptance and stability in different solvents are required. In conclusion, this study demonstrates that ancestral sequence reconstruction is a protein engineering technique effective in enhancing protein thermostability and should be considered a more viable alternative to directed evolution and rational design.

Alcohol dehydrogenases (ADH) are important tools for generating chiral α-hydroxyketones. Previously, only the ADH of Thauera aromatica was known to convert cyclic α-diketones with appropriate preference. Here, we extend the spectrum of suitable enzymes by three alcohol dehydrogenases from Citrifermentans bemidjiense (CibADH), Deferrisoma camini (DecADH), and Thauera phenylacetica (ThpADH). Of these, DecADH is characterized by very high thermostability; CibADH and ThpADH convert α-halogenated cyclohexanones with increased activity. Otherwise, however, the substrate spectrum of all four ADHs is highly conserved. Structural considerations led to the conclusion that conversion of diketones requires not only the expansion of the active site into a large binding pocket, but also the circumferential modification of almost all amino acid residues that form the first shell of the binding pocket. The constellation appears to be overall highly specific for the relative positioning of the carbonyl functions and the size of the C-ring.

Les amylosaccharases sont des α-transglucosylases catalysant naturellement la synthèse exclusive d’α-1,4-glucanes à partir du saccharose. Ces enzymes produisent également des composés secondaires et, en particulier, des isomères du saccharose tels que le turanose et le tréhalulose.L’objectif de cette thèse a consisté à utiliser un panel de techniques biophysiques et biochimiques afin d’étudier les amylosaccharases de Deinococcus geothermalis (ASDg) et Neisseria polysaccharea (ASNp) afin de comprendre les relations unissant la structure, la flexibilité et la fonction de ces enzymes.La première étude rapporte la caractérisation structurale et biophysique de l’amylosaccharase la plus thermostable connue à ce jour, l’amylosaccharase de Deinococcus geothermalis. La structure tridimensionnelle révèle une organisation dimérique en solution, jamais rapportée pour une amylosaccharase. Grâce à l’analyse de l’interface dimérique et à des travaux d’analyse de séquences, une séquence signature de dimérisation a été identifiée. En rigidifiant la structure de l’ASDg, la structure quaternaire contribue à l’augmentation de la stabilité thermique de la protéine. La spécificité de production des isomères du saccharose par les amylosaccharases a été étudiée. Les résultats décrivent, pour la première fois, les structures de l’ASDg et de l’ASNp en complexe avec le turanose. Dans l’ASNp, les résidus clefs forcent le résidu fructosyle à adopter une conformation linéaire positionnant idéalement le O3’ pour sa glucosylation expliquant la formation préférentielle de turanose par l’enzyme. Ces résidus sont absents ou placés différemment dans l’ASDg. En conséquence, l’ASDg lie principalement les formes furanoses du fructose avec un faible réseau d’interactions. La topologie du sous-site +1 permet donc différents modes de liaison du fructose en accord avec la capacité de l’ASDg à produire une plus grande quantité de tréhalulose par rapport à l’ASNp.Dans la seconde étude, des techniques de mutagenèse à saturation et combinatoire ciblées sur les acides aminés voisins du site actif ont été utilisées pour modifier la spécificité d'accepteur de l’ASNp. Le criblage de trois bibliothèques semi-rationnelles représentant un total de 20 000 variants a permis d’isoler trois doubles mutants montrant une amélioration spectaculaire de spécificité à la fois vis-à-vis du saccharose, le substrat donneur et de l’accepteur α-allyl-N-acetyl-2-désoxy-α-D-glucopyranoside par rapport au type sauvage de l’ASNp. De tels niveaux d'amélioration d'activité n'ont jamais été signalés auparavant pour cette classe d’enzymes actives sur les sucres. L’analyse par cristallographie des rayons X de la structure des meilleures enzymes mutantes suivie par des simulations de dynamique moléculaire ont montré une rigidité locale du sous-site -1 couplée à une flexibilité des boucles impliquées dans la topologie du site actif. Ces faits pourraient être à l’origine des performances catalytiques accrues de ces enzymes mutantes. L'étude démontre l'importance, lors de la conception des bibliothèques de variants, de tenir compte de la conformation locale des résidus catalytiques ainsi que de la dynamique des protéines au cours du processus catalytique / Amylosucrases are sucrose-utilizing α-transglucosylases that naturally catalyze the synthesis of α-glucans, exclusively linked through α-1,4 linkages. Side-products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes.The objective of this thesis concerned the application of biophysical and biochemical techniques to study amylosucrases from Deinococcus geothermalis (DgAS) and Neisseria polysaccharea (NpAS) in order to investigate relationships between structure, flexibility and function of these enzymes.In the first study, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis. The 3D-structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the DgAS and NpAS in complex with turanose. In NpAS, key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanose tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS.In the second study, iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of NpAS and sort out improved variants. From the screening of three semi-rational sub-libraries accounting in total for 20,000 variants, we report here the isolation of three double-mutants displaying a spectacular specificity enhancement towards both sucrose, the donor substrate, and the α-ally-N-acetyl-2-deoxy-α-D-glucopyranoside acceptor compared to wild-type N. polysaccharea amylosucrase. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structural analysis of the best performing enzymes followed by Molecular Dynamics simulations showed both local rigidity of the -1 subsite and flexibility of loops involved in active site topology which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance when designing enzyme libraries of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process