Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

生物－热催化过渡带沉积成岩演化及其成烃作用研究

The biothermocatalytic transitional zone gas is a new type of natural gas genetic theory, and also an clean, effective and high quality energy with shallow burial depth, wide distribution and few investment. Meanwhile, this puts biothermocatalytic transitional zone gas in important position to the energy resource and it is a challenging front study project. This paper introduces the concept, the present situation of study and developmental trend about biothermocatalytic transitional zone gas in detail. Then by using heat simulating of source rocks and catalysis mechanism analysis in the laboratory and studying structural evolution, sedimentation, diagenesis and the conditions of accumulation formation and so on, this paper also discusses catalytic mechanism and evolutionary model of the biothermocatalytic transitional zone gas formation, and establishes the methods of appraisal parameter and resources prediction about the biothermocatalytic transitional zone gas. At last, it shows that geochemical characteristics and differentiated mark of the biothermocatalytic transitional zone gas, and perfect natural gas genetic theory, and points out the conditions of accumulation formation, distribution characteristics and potential distribution region on the biothermocatalytic transitional zone gas m China. The paper mainly focuses on the formation mechanism and the resources potential about the biothermocatalytic transitional zone gas. Based on filed work, it is attached importance to a combination of macroscopic and microcosmic analysis, and the firsthand data are obtained to build up framework and model of the study by applying geologic theory. Based on sedimentary structure, it is expounded that structural actions have an effect on filling space and developmental cource of sediments and evolution of source rocks. Carried out sedimentary environment, sequence stratigraphy, sedimentary system and diagenesis and so on, it is concluded that diagenesis influences developmental evolution of source rocks, and basic geologic conditions of the biothermocatalytic transitional zone gas. Applying experiment simulating and catalytic simulating as well as chemical analysis, catalytic mechanism of clay minerals is discussed. Combined diagenecic dynamics with isotope fractionation dynamics, it is established that basis and method of resource appraisal about the biothermocatalytic transitional zone gas. All these results effectively assess and predict oil&gas resources about the biothermocatalytic transitional zone gas-bearing typical basin in China. I read more than 170 volumes on the biothermocatalytic transitional zone gas and complete the dissertation' summary with some 2.4 ten thousand words, draw up study contents in some detail and set up feasible experimental method and technologic course. 160 pieces of samples are obtained in oilfield such as Liaohe, Shengli, Dagang and Subei and so on, some 86 natural gas samples and more than 30 crude oil samples. Core profiles about 12 wells were observed and some 300 geologic photos were taken. Six papers were published in the center academic journal at home and abroad. Collected samples were analysised more than 1000 times, at last I complete this dissertation with more than 8 ten thousand words, and with 40 figures and 4 plates. According to these studies, it is concluded the following results and understandings. 1. The study indicates structural evolution and action of sedimentary basin influence and control the formation and accumulation the biothermocatalytic transitional zone gas. Then, the structural action can not only control accommodation space of sediments and the origin, migration and accumulation of hydrocarbon matters, but also can supply the origin of energy for hygrocarbon matters foramtion. 2. Sedimentary environments of the biothermocatalytic transitional zone gas are lake, river and swamp delta- alluvial fan sedimentary systems, having a warm, hot and humid climate. Fluctuation of lake level is from low to high., frequency, and piling rate of sedimentary center is high, which reflect a stable depression and rapidly filling sedimentary course, then resulting in source rocks with organic matter. 3. The paper perfects the natural gas genetic theory which is compound and continuous. It expounds the biothermocatalytic transitional zone gas is a special gas formation stage in continuous evolutionary sequence of organic matter, whose exogenic force is temperture and catalysis of clay minerals, at the same time, having decarbxylation, deamination and so on. 4. The methodology is established which is a combination of SEM, TEM and Engery spectrum analysis to identify microstructure of crystal morphology about clay minerals. Using differential thermal-chromatographic analysis, it can understand that hydrocarbon formation potential of different typies kerogens and catalytic method of all kinds of mineral matrix, and improve the surface acidity technology of clay minerals measured by the pyridine analytic method. 5. The experiments confirm catalysis of clay minerals to organic matter hygrocarbon formation. At low temperature (<300 ℃), there is mainly catalysis of montmorillonite, which can improve 2-3 times about produced gas of organic matters and the pyrolyzed temperature decreased 50 ℃; while at the high temperature, there is mainly catalysis of illite which can improve more than 2 times about produced gas of organic matters. 6. It is established the function relationship between organic matter (reactant) concentration and temperature, pressure, time, water and so on, that is C=f (D, t). Using Rali isotope fractionation effect to get methane isotope fractionation formula. According to the relationship between isotope fractionation of diagenesis and depth, and combined with sedimentary rate of the region, it is estimated that relict gas of the biothermocatalytic transitional zone gas in the representative basin. 7. It is revealed that hydrocarbon formation mechanism of the biothermocatalytic transitional zone gas is mainly from montmorillonite to mixed minerals during diagenesis. In interlayer, a lot of Al~(3+) substitute for Si~(4+), resulting in a imbalance between surface charge and interlayer charge of clay minerals and the occurrence of the Lewis and Bronsted acid sites, which promote to form the carbon cation. The cation can form alkene or small carbon cation. 8. It is addressed the comprehensive identification mark of the biothermo - catalytic transitional zone gas. In the temproal-spatial' distribution, its source rocks is mainly Palaeogene, secondly Cretaceous and Jurassic of Mesozoic, Triassic, having mudy rocks and coal-rich, their organic carbon being 0.2% and 0.4% respectively. The vitrinite reflection factor in source rocks Ro is 0.3-0.65%, a few up to 0.2%. The burial depth is 1000-3000m, being characterized by emerge of itself, reservoir of itself, shallow burial depth. In the transitional zone, from shallow to deep, contents of montmorillonites are progressively reduced while contents of illites increasing. Under SEM, it is observed that montmorillonites change into illite.s, firstly being mixed illite/ montmorillonite with burr-like, then itlite with silk-like. Carbon isotope of methane in the biothermocatatytic transitional zone gas , namely δ~(13)C_1-45‰- -60 ‰. 9. From the evolutionary sequence of time, distribution of the biothermocatalytic transitional zone gas is mainly oil&gas bearing basin in the Mesozoic-Neozoic Era. From the distribution region, it is mainly eastern stuctural active region and three large depressions in Bohaiwang basin. But most of them are located in evolutionary stage of the transitional zone, having the better relationship between produced, reservoir and seal layers, which is favorable about forming the biothermocatalytic transitional zone gas reservoir, and finding large gas (oil) field.

New tools provide a second look at HDV ribozyme structure, dynamics and cleavage.

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions.

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: New insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily .

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range.The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg+2 binding first (Kd =140 ± 40 M), are kcat = 105 ± 2 s-1 and P-pyr Km = 5 ± 1 M. PEP (slow substrate kcat = 2 × 10-4 s-1), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 ± 0.1 mM, 17 ± 1 M, and 210 ± 10 M, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (/)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

Structure of the adenylation domain of an NAD+-dependent DNA ligase

Background: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD+4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target.

Results: The crystal structure of the adenylation domain of the NAD+-dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 Å resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD+-binding site.

Conclusions: Comparison of the structure of the NAD+4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide: oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

Development of Novel Activity-Based Probes for the Detection of Serine Proteases

Cystic Fibrosis (CF) is a genetic disease featuring a chronic cycle of inflammation and infection in the airways of sufferers. Mutations lead to altered ion transport, which in turn causes dehydrated airways and reduced mucociliary clearance which predisposes the patient to infection, resulting in a severe immune response and tissue destruction (1). Airway dehydration is primarily caused by the hyperabsorption of sodium by the epithelial sodium channel (ENaC) (2). ENaC is activated by the action of a number of predominantly trypsin-like Channel Activating Proteases (CAPs) including prostasin, matriptase and furin (3). Additional proteases known to activate ENaC include human airway trypsin (3), plasmin, neutrophil elastase and chymotrypsin (4).

Activity profiling is a valuable technique which involves the use of small inhibitory molecules called Activity-Based Probes (ABPs) which can be used to covalently label the active site of proteases and provide a range of information regarding its structure, catalytic mechanism, location and function within biological systems. The development of novel ABPs for CAPs, would enhance understanding of the role of these proteases in CF airways disease and in particular their role in ENaC activation and airway dehydration. This project investigates the application of a range of novel broad-spectrum ABPs targeting the various subclasses of serine proteases, to include those proteases involved in ENaC activation. Additionally, the application of more selective ABPs in detecting specific serine proteases is investigated.

Compounds were synthesised by Solid-Phase Peptide Synthesis (SPPS) using a standard Fmoc/tBu strategy. Kinetic evaluation of synthesised ABPs against various serine proteases was determined by fluorogenic steady-state enzyme assays. Furthermore, application of ABPs and confirmation of irreversible nature of the compounds was carried out through SDS-PAGE and electroblotting techniques.

Synthesised compounds showed potent irreversible inhibition of serine proteases within their respective targeting class (NAP855 vs Trypsin k3/Ki = 2.60 x 106 M-1 min-1, NFP849 vs Chymotrypsin k3/Ki = 1.28 x 106 M-1 min-1 and NVP800 vs Neutrophil Elastase k3/Ki = 6.41 x 104 M-1 min-1). Furthermore ABPs showed little to no cross-reactivity between classes and so display selectivity between classes. The irreversible nature of compounds was further demonstrated through labelling of proteases, followed by separation and detection via SDS-PAGE and electroblotting techniques. Targeted labelling of active proteases only, was demonstrated by failure of ABPs to detect previously inactivated proteases. Extension of the substrate recognition site within probes resulted in an increased potency and selectivity in the detection of the target proteases. Successful detection of neutrophil elastase from CF sputum samples by NVP800, demonstrated the application of compounds within biological samples and their potential use in identifying further proteases involved in ENaC activation and airway dehydration in CF patients.

Highly Electrocatalytic Activity of RuO2 Nanocrystals for Triiodide Reduction in Dye-Sensitized Solar Cells

Dye-sensitized solar cells (DSCs) are promising alternatives to conventional silicon devices because of their simple fabrication procedure, low cost, and high efficiency. Platinum is generally used as a superior counter electrode (CE) material, but the disadvantages such as high cost and low abundance greatly restrict the large-scale application of DSCs. An efficient and sustainable way to overcome the limited supply of Pt is the development of high-efficiency Pt-free CE materials, which should possess both high electrical conductivity and superior electrocatalytic activity simultaneously. Herein, for the first time, a two-step strategy to synthesize ruthenium dioxide (RuO2) nanocrystals is reported, and it is shown that RuO2 catalysts exhibit promising electrocatalytic activity towards triiodide reduction, which results in comparable energy conversion efficiency to that of conventional Pt CEs. More importantly, by virtue of first-principles calculations, the catalytic mechanism of electrocatalysis for triiodide reduction on various CEs is investigated systematically and it is found that the electrochemical triiodide reduction reaction on RuO2 catalyst surfaces can be enhanced significantly, owing to the ideal combination of good electrocatalytic activity and high electrical conductivity.

ArnT proteins that catalyse the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases

ArnT is a glycosyltransferase that catalyses the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyse lipid A modification with L-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

Galactokinase promiscuity: a question of flexibility?

Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering.

Carbapenemases of Serratia fonticola UTAD54

As carbapenemases, serínicas e metalo-β-lactamases (MBLs), formam um grupo cada vez mais importante de β-lactamases capazes de tornar as bactérias resistentes a antibióticos β-lactâmicos, incluindo carbapenemos utilizados como antibióticos de último recurso no tratamento de infecções causadas por bactérias multirresistentes. De modo a compreender melhor a relação estrutura-função deste grupo de enzimas, prosseguimos com a caracterização bioquímica e estrutural das carbapenemases SFC-1 e Sfh-I específicas de Serratia fonticola UTAD54, uma estirpe ambiental isolada previamente de águas de consumo não tratadas no Nordeste de Portugal. Ambas as β-lactamases foram sobre-expressas em Escherichia coli e purificadas por cromatografia líquida. A SFC-1 recombinante, uma carbapenemase serínica, hidrolisa eficientemente antibióticos β-lactâmicos de todas as classes e exibe, comparativamente a enzimas relacionadas (ex. KPC), uma maior eficiência contra a ceftazidima e uma menor susceptibilidade aos inibidores convencionais das β-lactamases. As estruturas do cristal da SFC-1 nativa e de complexos de mutantes, obtidos por mutagénese dirigida, com o meropenemo não hidrolisado e na forma de acetilenzima foram determinados por substituição molecular utilizando cristalografia de raios-X. A estrutura da SFC-1 contém todas as características conservadas do centro activo das carbapenemases de classe A. Nas estruturas dos mutantes o meropenemo aparece orientado no centro activo por Thr236 e Thr238, posicionando-o próximo da Ser130 para a transferência do protão. Nas enzimas de classe A inibidas por carbapenemos, a interacção com a Arg244 impõe uma orientação diferente do meropenemo ligado, prejudicando a transferência do protão. Estas constituem as primeiras estruturas de uma carbapenemase de classe A com um carbapenemo no centro activo e revelam que estas enzimas alteram a orientação do meropenemo ligado para promover a catálise, sem alteração significativa da estrutura geral. A Sfh-I, tal como as outras MBLs da subclasse B2, apresenta um perfil de substratos reduzido, que inclui maioritariamente os carbapenemos. A Sfh-I hidrolisa imipenemo e meropenemo com um kcat de 51 e 109 s-1 e um KM de 79 e 215 μM, respectivamente. A Sfh-I liga um equivalente de zinco, como demonstrado por espectrometria de massa. Contrariamente a enzimas da subclasse B2 previamente caracterizadas, a Sfh-I hidrolisa a cefepima, mostrando que a Sfh-I é uma MBL da subclasse B2 com propriedades únicas. Por espectroscopia de fluorescência mostrou-se que a Sfh-I é capaz de ligar até 3 equivalentes de zinco (Kd2 = 95 μM; Kd3 = 2.3 mM). A estrutura do cristal da Sfh-I, determinada por substituição molecular utilizando a CphA como modelo, é a primeira para uma MBL da subclasse B2 não ligada. Esta estrutura revela a disposição das moléculas de água no centro activo corroborando um mecanismo catalítico para as MBLs da subclasse B2 no qual a His118, em vez do Asp120 proposto anteriormente, activa a molécula de água nucleofílica.

Études Structurales par Résonance Magnétique Nucléaire (RMN) du Site Actif du Ribozyme VS de Neurospora.

Nous étudions le ribozyme VS de Neurospora, en tant que système modèle, pour augmenter nos connaissances sur la relation entre la structure et la fonction chez les ARNs, ainsi que pour mieux comprendre le mécanisme de clivage de ce ribozyme. Il a été proposé précédemment que la boucle interne A730 dans la tige-boucle VI (SLVI) contient le site actif du ribozyme et lie un ou plusieurs ions métalliques qui pourraient participer au mécanisme réactionnel. Nous avons déterminé par spectroscopie RMN la structure de la tige-boucle SLVI contenant la boucle A730 afin d’éclaircir ce mécanisme. La structure obtenue est en accord avec les études biochimiques antérieures et présente un ou plusieurs sites de liaison au magnésium associé à la boucle interne. Suite à des études de cinétique et de mutagenèse, il a été proposé qu’une adénine localisée dans le site actif, A756, participe à la catalyse par acide/base générale. Des études de pH effectuées précédemment ont identifié un pKa catalytique (5.2-5.8) qui correspond probablement à l’équilibre de protonation du A756. À l’aide de méthodes utilisant le carbone-13, nous avons identifié un pKa modifié appartenant au A756, ce qui supporte le rôle de ce résidu dans la catalyse par acide/base générale. Les études structurales présentées ici aident donc à augmenter notre compréhension du mécanisme de clivage chez le ribozyme VS.

Structure d'une tagatose-1,6-bisphosphate aldolase de classe I : étude d'une apparente perte de stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase de classe I qui fait montre d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-biphosphate (TBP), mais également du fructose-1,6-biphosphate (FBP), du sorbose-1,6-biphosphate et du psicose-1,6-biphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution de la structure native et en complexe avec le DHAP, a respectivement 1.87 et 1.92 Å de résolution. Ces mêmes structures ont permis de se représenter plus clairement le site actif de l'enzyme en général, et les résidus catalytiques en particulier. Le trempage des cristaux de TBP aldolase dans une solution saturante de DHAP a en outre permis de piéger un authentique intermédiaire iminium, ainsi que sa géométrie particulière en atteste. Des expériences d'échange de proton, entreprises afin d'évaluer le stéréoisomérisme du transfert de proton catalytique, ont également permis de faire une intéressante découverte : la TBP aldolase ne peut déprotoner le coté pro-R du C3 du DHAP, mais peut le protonner. Ce résultat, ainsi que la comparaison de la structure du complexe TBP aldolase-DHAP avec la structure du complexe FBP aldolase de muscle de lapin- DHAP, pointe vers un isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP. De plus, la résolution de ces deux structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.

Étude structure-fonction des fructose-1,6-bisphosphate aldolases métallo-dépendantes : mécanisme catalytique et développement d’antimicrobiens

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques.