Richard Daniellou

Unraveling the Substrate Recognition Mechanism and Specificity of the Unusual Glycosyl Hydrolase Family 29 BT2192 from Bacteroides thetaiotaomicron

Glycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining α-l-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak α-l-fucosidase and β-d-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-d-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate α-l-fucose from binding efficiently to the fucosidase-like active site relative to other GH29 enzymes. β-d-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewis(X) antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6-7 Å apart.

TLC-UV hyphenated with MALDI-TOFMS for the screening of invertase substrates in plant extracts

In this study, thin-layer chromatography (TLC) hyphenated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the screening of invertase substrates in complex matrices. BfrA, a specific β-D-fructofuranosidase from Leishmania major, was chosen as a model enzyme to screen biological activity in plant extracts due to its capacity to hydrolyze specific carbohydrates. TLC was considered to be a reliable technique for screening substrates (bioactive molecules) in plant extracts due to its quantitative capabilities whereas MALDI-TOFMS was particularly useful for rapid identification. The first part of this approach consisted of a differential analysis by TLC-densitometry to highlight band under- and over-expressions in plant extract between blank and enzymatic reaction. Zones of interest were then immediately analyzed by TLC-MALDI-TOFMS coupling to identify bioactive molecules. Development of the method presented various challenges: the separation and analysis of isomers (such as glucose and fructose), the high matrix effect (demonstrated by the analysis of products with invertase enzyme naturally present in plant extract), and the analysis of polar molecules with low molecular mass (sugars). Thanks to the separative technique, the specificity of detection, and the high precision of the characterization, this method was shown to be feasible for the analysis of bioactive molecules in complex mixtures containing interfering compounds (e.g. proteins, salts). Overall, this study demonstrates that Thin-layer chromatography hyphenated with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a simple, rapid, precise and efficient method for the analysis of suitable substrates in raw samples.

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism.

Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity

This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the β-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H2O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (KM; Vmax) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars.

Chapter 10:Enzymatic thioglycosylation: current knowledge and challenges

Carbohydrates play an important part in a vast array of biological processes and therefore glycomimetics are currently becoming a powerful class of novel therapeutics.1 Amongst them, thioglycosides, in which a sulfur atom has replaced the glycosidic oxygen atom, are tolerated by most biological systems. Their major advantages rely on the fact that they adopt similar conformations than the corresponding O-glycosides and especially that they prove to be less sensitive to acid/base or enzyme-mediated hydrolysis. Such compounds have already demonstrated to be valuable tools as good chemical donors for synthetic purposes,2,3 as stable intermediates in X-ray crystallographic analysis of proteins4 and, of particular interest, as competitive inhibitors of a wide range of glycosidases (or glycoside hydrolases, GH) involved in numerous diseases.5 Besides the synthetic methodologies developed throughout the years by organic chemists, the presence of natural S-glycosylconjugates was recently assessed and led to the discovery of the enzymes involved in such rare biocatalytic processes. In parallel, the increase of knowledge on the mechanism and the structure of glycosidases has conducted to the development of original catalyst with greatly improved synthetic properties for thioglycosidic linkages.

Enzymatic synthesis of glycosides: from natural O- and N-glycosides to rare C- and S-glycosides

Carbohydrate related enzymes, like glycosyltransferases and glycoside hydrolases, are nowadays more easily accessible and are thought to represent powerful and greener alternatives to conventional chemical glycosylation procedures. The knowledge of their corresponding mechanisms has already allowed the development of efficient biocatalysed syntheses of complex O-glycosides. These enzymes can also now be applied to the formation of rare or unnatural glycosidic linkages.

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry

Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

Chemo-enzymatic synthesis of 3-O- (β-d-glycopyranosyl)-sn-glycerols and their evaluation as preservative in cosmetics

Abstract d-Glycopyranosyl glycerols are common natural products and exhibit strong biological properties, notably as moisturizing agents in cosmetics. Their chemical synthesis remains tedious thus decreasing their potential industrial and economic development, as well as the study of their structure-function relationships. In this work, the chemo-enzymatic synthesis of three enantiopure 3-O-(β-d-glycopyranosyl)-sn-glycerols was efficiently performed using an original glycosidase from Dictyoglomus thermophilum and their preservatives properties were assessed using a challenge test method. Amongst them, the 3-O-(β-d-glucopyranosyl)-sn-glycerol exhibited a specific anti-fungus activity.

Low molecular weight carbohydrate-based hydrogelators

This review will focus on the ability of synthetic small (low-molecular weight) carbohydrates to form hydrogels and describe their key parameters.