Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum.

BACKGROUND: Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster. RESULTS: Genetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD'EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw. CONCLUSIONS: This study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid-Water Interfaces to Access Its Insoluble Substrate.

Fatty acid photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions, and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. The kinetics of adsorption onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with a higher affinity in the presence of FAs. The FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues about the mode of action of FAP and the development of FAP-based bioconversion processes.

Structural Contour Map of the Iota Carbonic Anhydrase from the Diatom Thalassiosira pseudonana Using a Multiprong Approach.

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3-. The "iota" class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium-calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat "drone-like" shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.

Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surface-coated substrate.

Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10 ng of enzymes, against 100 ng to 10 µg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2.

BACKGROUND: Heterodimeric phospholipase A2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity. METHODS: Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids. RESULTS: Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs. CONCLUSION: Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity. GENERAL SIGNIFICANCE: New informations are given to further establish the structure-function relationships of the Sm-PLGV.

The galactolipase activity of Fusarium solani (phospho)lipase.

The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658±146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785±83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991±85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.

Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells.

Carbon acquisition, assimilation and storage in eukaryotic microalgae and cyanobacteria occur in multiple compartments that have been characterised by the location of the enzymes involved in these functions. These compartments can be delimited by bilayer membranes, such as the chloroplast, the lumen, the peroxisome, the mitochondria or monolayer membranes, such as lipid droplets or plastoglobules. They can also originate from liquid-liquid phase separation such as the pyrenoid. Multiple exchanges exist between the intracellular microcompartments, and these are reviewed for the CO2 concentration mechanism, the Calvin-Benson-Bassham cycle, the lipid metabolism and the cellular energetic balance. Progress in microscopy and spectroscopic methods opens new perspectives to characterise the molecular consequences of the location of the proteins involved, including intrinsically disordered proteins.

Role of the Solute-Binding Protein CuaD in the Signaling and Regulating Pathway of Cellobiose and Cellulose Utilization in Ruminiclostridium cellulolyticum.

In Ruminiclostridium cellulolyticum, cellobiose is imported by the CuaABC ATP-binding cassette transporter containing the solute-binding protein (SBP) CuaA and is further degraded in the cytosol by the cellobiose phosphorylase CbpA. The genes encoding these proteins have been shown to be essential for cellobiose and cellulose utilization. Here, we show that a second SBP (CuaD), whose gene is adjacent to two genes encoding a putative two-component regulation system (CuaSR), forms a three-component system with CuaS and CuaR. Studies of mutant and recombinant strains of R. cellulolyticum have indicated that cuaD is important for the growth of strains on cellobiose and cellulose. Furthermore, the results of our RT-qPCR experiments suggest that both the three (CuaDSR)- and the two (CuaSR)-component systems are able to perceive the cellobiose signal. However, the strain producing the three-component system is more efficient in its cellobiose and cellulose utilization. As CuaD binds to CuaS, we propose an in-silico model of the complex made up of two extracellular domains of CuaS and two of CuaD. CuaD allows microorganisms to detect very low concentrations of cellobiose due to its high affinity and specificity for this disaccharide, and together with CuaSR, it triggers the expression of the cuaABC-cbpA genes involved in cellodextrins uptake.

The structure of human DNase I bound to magnesium and phosphate ions points to a catalytic mechanism common to members of the DNase I-like superfamily.

Recombinant human DNase I (Pulmozyme, dornase alfa) is used for the treatment of cystic fibrosis where it improves lung function and reduces the number of exacerbations. The physiological mechanism of action is thought to involve the reduction of the viscoelasticity of cystic fibrosis sputum by hydrolyzing high concentrations of DNA into low-molecular mass fragments. Here we describe the 1.95 Å resolution crystal structure of recombinant human DNase I (rhDNase I) in complex with magnesium and phosphate ions, both bound in the active site. Complementary mutagenesis data of rhDNase I coupled to a comprehensive structural analysis of the DNase I-like superfamily argue for the key catalytic role of Asn7, which is invariant among mammalian DNase I enzymes and members of this superfamily, through stabilization of the magnesium ion coordination sphere. Overall, our combined structural and mutagenesis data suggest the occurrence of a magnesium-assisted pentavalent phosphate transition state in human DNase I during catalysis, where Asp168 may play a key role as a general catalytic base.

Direct analysis of phycobilisomal antenna proteins and metabolites in small cyanobacterial populations by laser ablation electrospray ionization mass spectrometry.

Due to their significance in energy and environmental and natural product research, as well as their large genetic diversity, rapid in situ analysis of cyanobacteria is of increasing interest. Metabolic profiles and the composition of energy harvesting antenna protein complexes are needed to understand how environmental factors affect the functioning of these microorganisms. Here, we show that laser ablation electrospray ionization (LAESI) mass spectrometry enables the direct analysis of phycobilisomal antenna proteins and report on numerous metabolites from intact cyanobacteria. Small populations (n < 616 ± 76) of vegetative Anabaena sp. PCC7120 cyanobacterial cells are analyzed by LAESI mass spectrometry. The spectra reveal the ratio of phycocyanin (C-PC) and allophycocyanin (APC) in the antenna complex, the subunit composition of the phycobiliproteins, and the tentative identity of over 30 metabolites and lipids. Metabolites are tentatively identified by accurate mass measurements, isotope distribution patterns, and literature searches. The rapid simultaneous analysis of abundant proteins and diverse metabolites enables the evaluation of the environmental response and metabolic adaptation of cyanobacteria and other microorganisms.

Intestinal phospholipase A2 from Sparidae species: Functional properties and cytotoxic potential evaluation.

Marine species have gained significant attention as potential source for a broad spectrum of bioactive proteins. Fish phospholipases A2 (PLA2) have attracted renewed interest due to their excellent properties in lipid digestion. Herein, we report for the first time the catalytic properties of two intestinal secreted PLA2 (sPLA2) identified from Diplodus sargus (IDsPLA2) and Sparus aurata (ISaPLA2). The highest sequence identity was obtained with recently isolated Sparidae digestive PLA2 (45%) and Human pancreatic PLA2 (42%). IDsPLA2 and ISaPLA2 were overexpressed in E. coli as inclusion bodies, refolded and purified. Both enzymes have improved thermostability compared to mammalian pancreatic sPLA2 since they are active and stable at 55 °C, with specific activities of 320 and 190 U mg-1 measured on phosphatidylcholine, respectively. Interestingly, IDsPLA2, but not ISaPLA2, revealed weak toxicity towards macrophages and suggests its involvement in cell membrane degradation. ISaPLA2 was found to be more active than IDsPLA2 when using the monolayer technique at 20 mN m-1. Structural models of both enzymes revealed their differences. In silico docking of phospholipids with both models allowed proposing key amino-acids in substrate binding and selectivity. Overall, these results provide insight into the enzymatic and structural properties of two novel sPLA2 with potential for future applications.

The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential α-linolenic acid.

Galactolipids, mainly monogalactosyl diglycerides and digalactosyl diglycerides are the main lipids found in the membranes of plants, algae and photosynthetic microorganisms like microalgae and cyanobacteria. As such, they are the main lipids present at the surface of earth. They may represent up to 80% of the fatty acid stocks, including a large proportion of polyunsaturated fatty acids mainly α-linolenic acid (ALA). Nevertheless, the interest in these lipids for nutrition and other applications remains overlooked, probably because they are dispersed in the biomass and are not as easy to extract as vegetable oils from oleaginous fruit and oil seeds. Another reason is that galactolipids only represent a small fraction of the acylglycerolipids present in modern human diet. In herbivores such as horses, fish and folivorous insects, galactolipids may however represent the main source of dietary fatty acids due to their dietary habits and digestion physiology. The development of galactolipase assays has led to the identification and characterization of the enzymes involved in the digestion of galactolipids in the gastrointestinal tract, as well as by microorganisms. Pancreatic lipase-related protein 2 (PLRP2) has been identified as an important factor of galactolipid digestion in humans, together with pancreatic carboxyl ester hydrolase (CEH). The levels of PLRP2 are particularly high in monogastric herbivores thus highlighting the peculiar role of PLRP2 in the digestion of plant lipids. Similarly, pancreatic lipase homologs are found to be expressed in the midgut of folivorous insects, in which a high galactolipase activity can be measured. In fish, however, CEH is the main galactolipase involved. This review discusses the origins and fatty acid composition of galactolipids and the physiological contribution of galactolipid digestion in various species. This overlooked aspect of lipid digestion ensures not only the intake of ALA from its main natural source, but also the main lipid source of energy for growth of some herbivorous species.

In vitro and in vivo exploration of the cellobiose and cellodextrin phosphorylases panel in Ruminiclostridium cellulolyticum: implication for cellulose catabolism.

BACKGROUND: In anaerobic cellulolytic micro-organisms, cellulolysis results in the action of several cellulases gathered in extracellular multi-enzyme complexes called cellulosomes. Their action releases cellobiose and longer cellodextrins which are imported and further degraded in the cytosol to fuel the cells. In Ruminiclostridium cellulolyticum, an anaerobic and cellulolytic mesophilic bacteria, three cellodextrin phosphorylases named CdpA, CdpB, and CdpC, were identified in addition to the cellobiose phosphorylase (CbpA) previously characterized. The present study aimed at characterizing them, exploring their implication during growth on cellulose to better understand the life-style of cellulolytic bacteria on such substrate. RESULTS: The three cellodextrin phosphorylases from R. cellulolyticum displayed marked different enzymatic characteristics. They are specific for cellodextrins of different lengths and present different k cat values. CdpC is the most active enzyme before CdpA, and CdpB is weakly active. Modeling studies revealed that a mutation of a conserved histidine residue in the phosphate ion-binding pocket in CdpB and CdpC might explain their activity-level differences. The genes encoding these enzymes are scattered over the chromosome of R. cellulolyticum and only the expression of the gene encoding the cellobiose phosphorylase and the gene cdpA is induced during cellulose growth. Characterization of four independent mutants constructed in R. cellulolyticum for each of the cellobiose and cellodextrin phosphorylases encoding genes indicated that only the cellobiose phosphorylase is essential for growth on cellulose. CONCLUSIONS: Unexpectedly, the cellobiose phosphorylase but not the cellodextrin phosphorylases is essential for the growth of the model bacterium on cellulose. This suggests that the bacterium adopts a "short" dextrin strategy to grow on cellulose, even though the use of long cellodextrins might be more energy-saving. Our results suggest marked differences in the cellulose catabolism developed among cellulolytic bacteria, which is a result that might impact the design of future engineered strains for biomass-to-biofuel conversion.

Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling.

The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906bp encoding a 301-amino acid polypeptide with a molecular mass of 30kDa. Based on sequence similarity with other fungal lipases, FSL2 contains a catalytic triad, consisting of Ser144, Asp198, and His256. FSL2 cDNA was subcloned into the pGAPZαA vector containing the Saccharomyces cerevisiae α-factor signal sequence and this construct was used to transform Pichia pastoris and achieve a high-level extracellular production of a FSL2 lipase. Maximum lipase activity was observed after 48h. The optimum activity of the purified recombinant enzyme was measured at pH 8.0-9.0 and 37°C. FSL2 is remarkably stable at alkaline pH values up to 12 and at temperatures below 40°C. It has high catalytic efficiency towards triglycerides with short to long chain fatty acids but with a marked preference for medium and long chain fatty acids. FSL2 activity is decreased at sodium taurodeoxycholate concentrations above the Critical Micelle Concentration (CMC) of this anionic detergent. However, lipase activity is enhanced by Ca2+ and inhibited by EDTA or Cu2+ and partially by Mg2+ or K+. In silico docking of medium chain triglycerides, monogalctolipids (MGDG), digalactolipids (DGDG) and long chain phospholipids in the active site of FSL2 reveals structural solutions.

Respective importance of protein folding and glycosylation in the thermal stability of recombinant feruloyl esterase A.

The thermal stability of four molecular forms (native, refolded, glycosylated, non-glycosylated) of feruloyl esterase A (FAEA) was studied. From the most to the least thermo-resistant, the four molecular species ranked as follows: (i) glycosylated form produced native, (ii) non-glycosylated form produced native, (iii) non-glycosylated form produced as inclusion bodies and refolded, and (iv) glycosylated form produced native chemically denatured and then refolded. On the basis of these results and of crystal structure data, we discuss the respective importance of protein folding and glycosylation in the thermal stability of recombinant FAEA.

Effects of the propeptide of group X secreted phospholipase A(2) on substrate specificity and interfacial activity on phospholipid monolayers.

Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance.

New insights into the pH-dependent interfacial adsorption of dog gastric lipase using the monolayer technique.

The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of interfacial enzymology and lipase-lipid interactions. The interfacial adsorption of dog gastric lipase (DGL) was monitored as a function of pH and surface pressure (Π), independently from the catalytic activity, using non-hydrolysable 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monomolecular films. The acid-stable DGL, which initiates fat digestion in the stomach, was then selected because its adsorption kinetics onto hydrophobic solid surfaces were already studied. This gastric lipase was therefore used as a model enzyme to validate both experimental and theoretical approaches. Results show that the adsorption process of DGL at the lipid/water interface depends on a pH-dependent adsorption equilibrium coefficient which is optimum at pH 5.0 (K(Ads) = 1.7 ± 0.05 × 10(8)M(-1)). KAds values further allowed an indirect estimation of the molar fraction (ΦE*(%), mol%) as well as the molecular area (AE*) of DGL adsorbed onto DLPC monolayer. Based on these data, a model for DGL adsorption onto DLPC monolayer at pH 5.0 is proposed for a surface pressure range of 15-25 mNm(-1).

Analysis of the discriminative inhibition of mammalian digestive lipases by 3-phenyl substituted 1,3,4-oxadiazol-2(3H)-ones.

We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e.MmPPOX and MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human (HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipase-related proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts (PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of DGL, we demonstrated that the phenoxy group plays a key role in specific molecular interactions within the lipase's active site. The absence of this group in the case of MPOX, as well as its connectivity to the neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory efficiency of these oxadiazolones and leads to a significant gain in selectivity towards the lipases tested. The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone derivatives could also provide useful leads for the development of novel and more discriminative inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal lipolysis process.

Structures of mutants of cellulase Cel48F of Clostridium cellulolyticum in complex with long hemithiocellooligosaccharides give rise to a new view of the substrate pathway during processive action.

An efficient breakdown of lignocellulosic biomass is a prerequisite for the production of second-generation biofuels. Cellulases are key enzymes in this process. We crystallized complexes between hemithio-cello-deca and dodecaoses and the inactive mutants E44Q and E55Q of the endo-processive cellulase Cel48F, one of the most abundant cellulases in cellulosomes from Clostridium cellulolyticum, to elucidate its processive mechanism. In both complexes, the cellooligosaccharides occupy similar positions in the tunnel part of the active site but are more or less buried into the cleft, which hosts the active site. In the E44Q complex, it proceeds along the upper part of the cavity, while it occupies in the E55Q complex the same productive binding subsites in the lower part of the cavity that have previously been reported in Cel48F/cellooligosaccharide complexes. In both cases, the sugar moieties are stabilized by stacking interactions with aromatic side chains and H bonds. The upper pathway is gated by Tyr403, which blocks its access in the E55Q complex and offers a new stacking interaction in the E44Q complex. The new structural data give rise to the hypothesis of a two-step mechanism in which processive action and chain disruption occupy different subsites at the end of their trajectory. In the first part of the mechanism, the chain may smoothly slide up to the leaving group site along the upper pathway, while in the second part, the chain is cleaved in the already described productive binding position located in the lower pathway. The solved native structure of Cel48F without any bound sugar in the active site confirms the two side-chain orientations of the proton donor Glu55 as observed in the complex structures.