Using Glucan Water Dikinase for in vitro glucan phosphorylation

Please click Additional Files below to see the full abstrac

Dédoublement enzymatique d'acides alpha-halogéno aryl acétique (Approche expérimentale et modélisation moléculaire)

L'étude a porté sur le dédoublement d'acides alpha-halogéno aryl acétique catalysé par les lipases. Ces composés sont utilisés pour la synthèse de molécules pharmaceutiques, ou autres, optiquement pures. Dans un premier temps, plusieurs lipases (RML, PCL, CALB, CRL, HLL) ont été testées pour leur capacité à dédoubler l'alpha-bromo-o-tolyl acétate d'éthyle en réaction de transestérification avec l'octanol. Une énantiosélectivité maximale de E=11,3 a été obtenue avec la lipase de R. miehei immobilisée sur polypropylène, laquelle est plus sélective que la lipase libre. D'autre part, la lipase de P. cepacia s'est révélée être beaucoup plus énantiosélective pour les substrats portant la substitution méthyle en position méta et para sur le cycle aromatique (E>50). Dans un deuxième temps, parmi tous les paramètres susceptibles de modifier l'énantiosélectivité de la lipase de P. cepacia pour le dédoublement de l'alpha-bromo phényl acétate d'éthyle, nous avons mis en évidence que la sélectivité de la lipase était fortement influencée par la nature de l'halogène du centre asymétrique ainsi que par la nature nucléofuge de la partie alkyle du substrat. L'immobilisation du biocatalyseur modifie également la sélectivité de la lipase. Enfin, par modélisation moléculaire, nous avons identifié les processus moléculaires responsables de la sélectivité de la lipase de P. cepacia pour l'énantiomère (R) de l'alpha-bromo phényl acétate d'éthyle (E=57). L'approche que nous avons suivie n'a jamais été décrite dans la littérature. Pour la première fois, en retraçant les trajectoires de chaque énantiomère dans le site actif de la lipase et sur la base des énergies d'interaction enzyme/substrat, nous avons montré que le site actif de l'enzyme est moins accessible à l'énantiomère (S) qu'à l'énantiomère (R). Un réseau d'acides aminés hydrophobes à chaînes latérales pivotantes (Val, Leu), tapissant les parois du site actif, semble intervenir dans le cheminement du substrat vers le site actif. En particulier, deux acides aminés Val-266 et Leu-17 forment un goulot d'étranglement qui semble intervenir dans la discrimination des énantiomères. La détermination de l'énantiosélectivité du mutant V266L porteur à la position 266 d'une chaîne latérale plus encombrante a confirmé ce résultat. En effet, pour ce mutant, la taille du goulot d'étranglement est réduite et l'énantiosélectivité est supérieure à 200.This study focused on lipase-catalysed resolution of alpha-halogeno aryl acetic acids. These compounds are used for the synthesis of enantiomerically pure pharmaceutical molecules. Several lipases (RML, PCL, CALB, CRL, HLL) were screened for their ability to catalyse the enantioselective transesterification of alpha-bromo-o-tolyl ethyl acetate with n-octanol. The best enantioselectivity was obtained with R. miehei lipase immobilized on polypropylene (E=11.3), which was more stereoselective than the free one. On the other hand, P. cepacia lipase was more enantioselective for the meta- and para-tolyl substrates (E>50). Among all the factors susceptible to modify the P. cepacia lipase enantioselectivity for the resolution of alpha-bromo phenyl ethyl acetate, we showed that lipase selectivity was dramatically influenced by the halogen at the stereo-centre and by the leaving alcohol constituting the alkyl part of the substrate. The immobilization of the biocatalyst also modifies the selectivity of lipases. Finally, by molecular modelling, we attempted to identify the molecular processes responsible for P. cepacia lipase selectivity for (R)-alpha-bromo phenyl ethyl acetate (E=57). The approach used, had never been described in the literature. For the first time, the trajectory of each enantiomer to the active site was mapped and the energy of enzyme/substrate interactions was calculated along the path. On the basis of interaction energy, we showed that the enzyme active site is less accessible for the S-enantiomer than for the R-one. A hydrophobic network of amino acids with pivoting side chains (Val, Leu), covering the sides of the active site, seems to play a role in driving the substrate to the active site. In particular, two amino acids Val-266 and Leu-17 form a bottleneck. We suggest that this structural fracture influences the discrimination of R,S-enantiomers. The determination of the enantioselectivity of the mutant V266L with a side chain more bulky at this position, supported this assumption. In fact, for this mutant, the size of the bottleneck is reduced, and the enantioselectivity was found to be higher than 200TOULOUSE-INSA (315552106) / SudocSudocFranceF

Natural and engineered transglycosylases: Green tools for the enzyme-based synthesis of glycoproducts

International audienceAn increasing number of transglycosylase-based processes provide access to oligosaccharides or glycoconjugates, some of them reaching performance levels compatible with industrial developments. Nevertheless, the full potential of transglycosylases has not been explored because of the challenges in transforming a glycoside hydrolase into an efficient transglycosylase. Advances in studying enzyme structure/function relationships, screening enzyme activity, and generating synthetic libraries guided by computational protein design or machine learning methods should considerably accelerate the development of these catalysts. The time has now come for researchers to uncover their possibilities and learn how to design and precisely refine their activity to respond more rapidly to the growing demand for well-defined glycosidic structures

Probing Substrate Promiscuity of Amylosucrase from Neisseria polysaccharea

The amylosucrase from Neisseria polysaccharea (NpAS) naturally catalyzes the synthesis of a variety of products from sucrose and shows signs of plasticity of its active site. p-nitrophenyl-alpha D-glucopyranoside was used by the wild-type enzyme, and this underlines the high specificity of the -1 subsite of NpAS for glucosyl donor substrates. D- and L-monosaccharides as well as polyols. With the exception of one compound, all were successfully glucosylated, and this showcases the tremendous plasticity of the +1 subsite of NpAS, which is responsible for acceptor recognition. The products obtained from the transglucosylation reactions of three selected acceptors were characterized, and they revealed original structures and enzyme enantiopreference, which were more particularly analyzed by in silico docking analyses

Laccases from Marine Organisms and Their Applications in the Biodegradation of Toxic and Environmental Pollutants: a Review

The discharge of industrial effluent creates environmental problems around the world and so necessitates the need for the economically expensive and sometimes technically problematic treatment of the wastewater. Laccases have enormous potential for the oxidative bioremediation of toxic xenobiotic compounds using only molecular oxygen as the sole cofactor for their reaction, and their application is regarded as environmentally friendly. Due to the low substrate specificity of laccases, they can oxidize a variety of substrates. Moreover, by using appropriate mediators, laccases can degrade a wide range of substrates, including those with structural complexity. Thus, laccases are an attractive alternative for wastewater treatment. Marine environments are rich in microorganisms that are exposed to extreme conditions, such as salinity, temperature, and pressure. Laccases from these microorganisms potentially have suitable properties that might be adaptive to bioremediation processes. This review provides the latest information on laccases from marine environments, their sources, biochemical properties, media composition for laccase production, and their applications in the bioremediation of industrial waste, especially focusing on dye decolorization

New efficient lipase from Yarrowia lipolytica for the resolution of 2-bromo-arylacetic acid esters

International audienceA new extracellular lipase (Lip2p) from the yeast Yarrowia lipolytica was used for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates for the pharmaceutical industry. Its efficiency for the transesterification of racemic mixtures with 1-octanol in n-octane was compared with the most efficient lipases described to date, lipases from Burkholderia cepacia and Rhizomucor miehei. Resolution of 2-bromo-p-tolylacetic acid ethyl ester catalyzed by Y. lipolytica lipase showed an enantio preference of 28, almost equal to that obtained with B. cepacia lipase (E = 30). Moreover, Y lipolytica lipase presents a higher catalytic activity and an (S)-enantiopreference, while B. cepacia lipase is (R)-enantiomer selective. The most interesting result is that Y lipolytica lipase has until now been the only enzyme able to catalyze the resolution of 2-bromo-o-tolylacetic acid ethyl ester (E = 27)

Alteration of enzyme activity and enantioselectivity by biomimetic encapsulation in silica particles

Direct encapsulation of esterase or lipase fused with the silica-precipitating R5 peptide from Cylindrotheca fusiformis in silica particles afforded high yields of active entrapped protein. The hydrolytic activity of both enzymes against p-nitrophenyl butyrate was similarly affected by encapsulation and the enantioselectivity of the esterase was both improved and inverted

A path planning approach for computing large-amplitude motions of flexible molecules

International audienceMotivation: Motion is inherent in molecular interactions. Molecular flexibility must be taken into account in order to develop accurate computational techniques for predicting interactions. Energy-based methods currently used in molecular modeling (i.e. molecular dynamics, Monte Carlo algorithms) are, in practice, only able to compute local motions while accounting for molecular flexibility. However, large-amplitude motions often occur in biological processes. We investigate the application of geometric path planning algorithms to compute such large motions in flexible molecular models. Our purpose is to exploit the efficacy of a geometric conformational search as a filtering stage before subsequent energy refinements. Results: In this paper two kinds of large-amplitude motion are treated: protein loop conformational changes (involving protein backbone flexibility) and ligand trajectories to deep active sites in proteins (involving ligand and protein side-chain flexibility). First studies performed using our two-stage approach (geometric search followed by energy refinements) show that, compared to classical molecular modeling methods, quite similar results can be obtained with a performance gain of several orders of magnitude. Furthermore, our results also indicate that the geometric stage can provide highly valuable information to biologists. Availability: The algorithms have been implemented in the general-purpose motion planning software Move3D, developed at LAAS-CNRS. We are currently working on an optimized stand-alone library that will be available to the scientific community

Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans

International audienceCarbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerousbiological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by theanomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing indemand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic andchemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD−MS methods for the precise structuralcharacterization of complex glycans