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International audience; 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. Adsorption kinetics onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with higher affinity in the presence of FAs. 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 for the mode of action of FAP and the development of FAP-based bioconversion processes.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviours and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel secreted biosurfactant polysaccharide (BPS) is temporally and spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus-inhibited acidic polymer built of randomly-acetylated β-linked tetrasaccharide repeats. Both BPS and the “shared good” EPS are produced by dedicated Wzx/Wzy-dependent polysaccharide assembly pathways distinct from that responsible for spore coat assembly. To our knowledge, such pathways have never-before been explicitly shown to synthesize a biosurfactant. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviours coordinating bacterial multicellularity.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated β-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity., A study of the social bacterium Myxococcus xanthus reveals that the bacteria preferentially secrete specific polysaccharides within distinct zones of a swarm to facilitate spreading across a surface.

International audience; A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M.tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.

Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 μM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects., Author summary Buruli ulcer is a necrotizing skin disease caused by an environmental mycobacterial pathogen. The pathogenesis of this neglected tropical disease involves the production of a toxin, mycolactone, which spreads through the tissues, away from the infecting organisms. Mycolactone has pleiotropic effects on fundamental cellular processes, resulting in pronounced cytotoxicity and immunosuppressive effects that together drive progressive ulceration. The molecular mechanisms underlying its cellular effects have been partly deciphered, but multiple cellular targets have been identified. A literature survey revealed that most of the identified targets of mycolactone are membrane receptors residing in particular domains of the plasma membrane. Despite its lipid-like nature, mycolactone has been shown to be intracellular, implying that it can cross the plasma membrane. We describe here a surprising membrane-reshaping effect of mycolactone due to effects on lipid domain formation. By reversing lateral lipid segregation, mycolactone may disrupt the formation of domains with well-established roles in the regulation of cellular signaling pathways. This remodeling of the cell plasma membrane may underlie the molecular events enabling mycolactone to attack multiple targets.

Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (NTAIL) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of NTAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. NTAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, NTAIL shows a much higher molecular area and forms a highly viscoelastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the NTAIL protein that folds into an antiparallel β-sheet at the A/W interface and presents strong intermolecular interactions.

The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) is assumed to be a good drug candidate for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. Understanding and improving its biochemical properties are essential for its oral administration. YLLIP2 is a highly glycosylated protein, with glycan chains accounting for about 13% of the molecular mass of the native protein. Two potential N-glycosylation sites (N113IS and N134NT) can be identified from YLLIP2 amino acid sequence. YLLIP2 mutants with single (N113Q or N134Q) or combined (N113Q/N134Q) substitutions of these glycosylation sites were expressed in the yeast Pichia pastoris, purified and characterized. Lipase specific activity and adsorption at the lipid–water interface were found to be decreased in the absence of N-glycosylation. It was thus shown that the glycosylated enzyme had a better ability to bind and penetrate a DLPC monolayer than the non-glycosylated N113Q/N134Q mutant. Comparison of wild-type glycosylated and non-glycosylated YLLIP2 shows that the N-glycosylation clearly contributes to the high stability of YLLIP2 in the presence of pepsin in vitro, and to a lower extent in the presence of chymotrypsin. The X-ray structure of the YLLIP2 N113Q/N134Q double mutant was obtained at 2.6 A resolution and was found to be identical to that of wild-type YLLIP2, with the lid in a closed conformation. Glycosylation is therefore not essential for a proper folding of YLLIP2. Practical applications: The LIP2 lipase from the yeast Yarrowia lipolytica is one of the most active lipases identified so far. Among the various applications envisioned for this enzyme, it seems particularly well adapted for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. It is active and stable at low pH values, resistant to bile salts, and its glycosylation allows a high resistance to pepsin. All these properties are important for developing the oral administration of digestive enzymes used as drugs.

The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of the overall catalytic process carried out by these interfacial enzymes. Gastric lipase, for instance, shows an apparent optimum activity on triglycerides (TAG) at acidic pH, which is controlled by its pH-dependent adsorption at lipid-water interfaces. Since gastric lipase acts on TAG droplets covered by phospholipids, but does not hydrolyze these lipids, phospholipid monolayers spread at the air-water interfaces can be used as biomimetic interfaces to study lipase adsorption and penetration through the phospholipid layer, independently from the catalytic activity. The adsorption of recombinant dog gastric lipase (rDGL) onto 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monolayers can be monitored by surface tensiometry at various enzyme concentrations, pHs, and surface pressures (Π). These experimental data and the use of Langmuir adsorption isotherm and Verger-de Haas' lipase kinetics models further allow estimating various parameters including the adsorption equilibrium constant (K

International audience; The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of the overall catalytic process carried out by these interfacial enzymes. Gastric lipase, for instance, shows an apparent optimum activity on triglycerides (TAG) at acidic pH, which is controlled by its pH-dependent adsorption at lipid–water interfaces. Since gastric lipase acts on TAG droplets covered by phospholipids, but does not hydrolyze these lipids, phospholipid monolayers spread at the air–water interfaces can be used as biomimetic interfaces to study lipase adsorption and penetration through the phospholipid layer, independently from the catalytic activity. The adsorption of recombinant dog gastric lipase (rDGL) onto 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monolayers can be monitored by surface tensiometry at various enzyme concentrations, pHs, and surface pressures (Π). These experimental data and the use of Langmuir adsorption isotherm and Verger–de Haas’ lipase kinetics models further allow estimating various parameters including the adsorption equilibrium constant (KAds), the interfacial concentration Γ E * , the molar fraction Φ E * % , mol % (ΦE*(%), mol%), and the molecular area A E * of rDGL adsorbed onto the DLPC monolayer under various conditions. Additional insight into rDGL adsorption/insertion on phospholipid monolayers can be obtained by combining ellipsometry, Langmuir–Blodgett film transfer, and atomic force microscopy. When using multicomponent phospholipid monolayers with phase separation, these techniques allow to visualizing how rDGL preferentially partitions toward liquid expanded phase and at phase boundaries, gets adsorbed at various levels of insertion and impacts on the lateral organization of lipids.

International audience; Intrinsically disordered proteins (IDPs) lack stable secondary and tertiary structure under physiological conditions in the absence of their biological partners and thus exist as dynamic ensembles of interconverting conformers, often highly soluble in water. However, in some cases, IDPs such as the ones involved in neurodegenerative diseases can form protein aggregates and their aggregation process may be triggered by the interaction with membranes. Although the interfacial behavior of globular proteins has been extensively studied, experimental data on IDPs at the air/water (A/W) and water/lipid interfaces are scarce. We studied here the intrinsically disordered C-terminal domain of the Hendra virus nucleoprotein (N TAIL) and compared its interfacial properties to those of lysozyme that is taken as a model globular protein of similar molecular mass. Adsorption of N TAIL at the A/W interface was studied in the absence and presence of phospholipids using Langmuir films, polarization modulated-infrared reflection-absorption spectroscopy, and an automated drop tensiometer for interfacial tension and elastic modulus determination with oscillating bubbles. N TAIL showed a significant surface activity, with a higher adsorption capacity at the A/W interface and penetration into egg phosphatidylcholine monolayer compared to lysozyme. Whereas lysozyme remains folded upon compression of the protein layer at the A/W interface and shows a quasi-pure elastic behavior, N TAIL shows a much higher molecular area and forms a highly visco-elastic film with a high dilational modulus. To our knowledge, a new disorder-to-order transition is thus observed for the N TAIL protein that folds into an antiparallel b-sheet at the A/W interface and presents strong intermolecular interactions.

International audience; Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity".

Owing to the large panel of biological functions of peptides and their high specificity and potency, the development of peptide-based therapeutic and diagnostic tools has received increasing interest. Peptide amphiphiles (PAs) are an emerging class of molecules in which a bioactive peptide is covalently conjugated to a hydrophobic moiety. Due to the coexistence in the molecule of a hydrophilic peptide sequence and a hydrophobic group, PAs are able to self-assemble spontaneously into a variety of nanostructures, such as monolayers, bilayers, and vesicles. In this work we have synthesized a disordered peptide, henceforth called R11, and two lipophilic derivatives of R11 bearing two alkyl chains, connected or not to R11 by an ethoxylic-based linker. The structural properties in solution of these new PAs were investigated using CD and NMR. R11 lipophilic derivatives display typical features of PAs, such as the formation of micelles and unilamellar vesicles. In addition, their surface properties were studied using Langmuir monomolecular films and the results obtained support the formation of molecular aggregates upon compression of the PA films. The presence of the alkyl chains induces not only the self-assembly of these new PAs into supramolecular aggregates but also a gain of structure within the disordered peptide.

Lipolytic activities of Yarrowia lipolytica LIP2 lipase (YLLIP2), human pancreatic (HPL) and dog gastric (DGL) lipases were first compared using lecithin-stabilized triacylglycerol (TAG) emulsions (Intralipid) at various pH and bile salt concentrations. Like DGL, YLLIP2 was able to hydrolyze TAG droplets covered by a lecithin monolayer, while HPL was not directly active on that substrate. These results were in good agreement with the respective kinetics of adsorption on phosphatidylcholine (PC) monomolecular films of the same three lipases, YLLIP2 being the most tensioactive lipase. YLLIP2 adsorption onto a PC monolayer spread at the air/water interface was influenced by pH-dependent changes in the enzyme/lipid interfacial association constant (KAds) which was optimum at pH 6.0 on long-chain egg PC monolayer, and at pH 5.0 on medium chain dilauroylphosphatidylcholine film. Using substrate monolayers (1,2-dicaprin, trioctanoin), YLLIP2 displayed the highest lipolytic activities on both substrates in the 25–35 mN m−1 surface pressure range. YLLIP2 was active in a large pH range and displayed a pH-dependent activity profile combining DGL and HPL features at pH values found in the stomach (pH 3–5) and in the intestine (pH 6–7), respectively. The apparent maximum activity of YLLIP2 was observed at acidic pH 4–6 and was therefore well correlated with an efficient interfacial binding at these pH levels, whatever the type of interfaces (Intralipid emulsions, substrate or PC monolayers). All these findings support the use of YLLIP2 in enzyme replacement therapy for the treatment of pancreatic exocrine insufficiency, a pathological situation in which an acidification of intestinal contents occurs.

CITREM is an emulsifier used in the food industry and contains citric acid esters of mono- and diglycerides (GCFE). It is generally recognized as safe but no publication on its digestibility under gastrointestinal conditions and impact on fat digestion was available. It was shown here that fatty acids are released from CITREM by gastric lipase, pancreatic lipase, pancreatic-lipase-related protein 2 and carboxyl ester hydrolase. A two-step in vitro digestion model mimicking lipolysis in the stomach and upper small intestine of term and preterm infants was then used to evaluate the digestibility of CITREM alone, CITREM-containing infant formula and fat emulsions, and isolated GCFE fractions. Overall, it was shown that fat digestion is not significantly changed by the presence of CITREM, and fatty acids contained in CITREM compounds are released to a large extent by lipases. Nevertheless, undigestible water-soluble compounds containing glycerol and citric acid units were identified, indicating that the ester bond between citric acid and glycerol is not fully hydrolyzed throughout the proposed digestion.

Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl d-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three d-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-d-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.

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).

International audience; The lipolysis reaction carried out by lipases at the water–lipid interface is a complex process including enzyme conformational changes, adsorption/desorption equilibrium and substrate hydrolysis. Mixed monomolecular films of the lipase inhibitor Orlistat and 1,2-dicaprin were used here to investigate the adsorption of dog gastric lipase (DGL) followed by the hydrolysis of 1,2-dicaprin. The combined study of these two essential catalysis steps was made possible thanks to the highest affinity of DGL for Orlistat than 1,2-dicaprin and the fact that the inhibition of DGL by Orlistat is reversible. Upon DGL binding to mixed 1,2-dicaprin/Orlistat monolayers, an increase in surface pressure reflecting lipase adsorption was first recorded. Limited amounts of Orlistat allowed to maintain DGL inactive on 1,2-dicaprin during a period of time that was sufficient to determine DGL adsorption and desorption rate constants. A decrease in surface pressure reflecting 1,2-dicaprin hydrolysis and product desorption was observed after the slow hydrolysis of the covalent DGL-Orlistat complex was complete. The rate of 1,2-dicaprin hydrolysis was recorded using the surface barostat technique. Based on a kinetic model describing the inhibition by Orlistat and the activity of DGL on a mixed 1,2-dicaprin/Orlistat monolayer spread at the air–water interface combined with surface pressure measurements, it was possible to monitor DGL adsorption at the lipid–water interface and substrate hydrolysis in the course of a single experiment. This allowed to assess the kcat/KM* ratio for DGL acting on 1,2-dicaprin monolayer, after showing that mixed monolayers containing a low fraction of Orlistat were similar to pure 1,2-dicaprin 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.