Jean-Marc Bonmatin

Recent trends in the biochemistry of surfactin

Abstract The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m−1 to 27 mN m−1 at a concentration as low as 20 μM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a β-hydroxy fatty acid with 13–15 C atoms. In solution, the molecule exhibits a characteristic “horse saddle” conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4′-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries.

Effects of neonicotinoids and fipronil on non-target invertebrates

We assessed the state of knowledge regarding the effects of large-scale pollution with neonicotinoid insecticides and fipronil on non-target invertebrate species of terrestrial, freshwater and marine environments. A large section of the assessment is dedicated to the state of knowledge on sublethal effects on honeybees (Apis mellifera) because this important pollinator is the most studied non-target invertebrate species. Lepidoptera (butterflies and moths), Lumbricidae (earthworms), Apoidae sensu lato (bumblebees, solitary bees) and the section “other invertebrates” review available studies on the other terrestrial species. The sections on freshwater and marine species are rather short as little is known so far about the impact of neonicotinoid insecticides and fipronil on the diverse invertebrate fauna of these widely exposed habitats. For terrestrial and aquatic invertebrate species, the known effects of neonicotinoid pesticides and fipronil are described ranging from organismal toxicology and behavioural effects to population-level effects. For earthworms, freshwater and marine species, the relation of findings to regulatory risk assessment is described. Neonicotinoid insecticides exhibit very high toxicity to a wide range of invertebrates, particularly insects, and field-realistic exposure is likely to result in both lethal and a broad range of important sublethal impacts. There is a major knowledge gap regarding impacts on the grand majority of invertebrates, many of which perform essential roles enabling healthy ecosystem functioning. The data on the few non-target species on which field tests have been performed are limited by major flaws in the outdated test protocols. Despite large knowledge gaps and uncertainties, enough knowledge exists to conclude that existing levels of pollution with neonicotinoids and fipronil resulting from presently authorized uses frequently exceed the lowest observed adverse effect concentrations and are thus likely to have large-scale and wide ranging negative biological and ecological impacts on a wide range of non-target invertebrates in terrestrial, aquatic, marine and benthic habitats.

Diversity Among Microbial Cyclic Lipopeptides: Iturins and Surfactins. Activity-Structure Relationships to Design New Bioactive Agents

A prominent group of bioactive lipopeptides produced by Bacillus species is constituted by iturins, surfactins and lichenysins. Interest in such substances results in their exceptional surfactant power, and their valuable antifungal, antibacterial, antitumoral and anti-Mycoplasma properties. As is typical for peptidic secondary-metabolites synthesized by the polyenzymic pathway, they are produced as mixtures of components varying in the peptidic and/or in the lipidic structure. In the context of structure-activity relationships, it is possible to take advantage of the adaptability of the biosynthesis system by systematically adding selected amino acids in the culture medium of the producing bacterium. When an amino acid is used as the sole nitrogen source, it is inserted directly into selected positions of the peptide sequence, thus amplifying the original structural microheterogeneity via a production of variants. This method revealed very efficient for increasing the amounts of preexisting variants and for building new variants of surfactins and lichenysins but totally inefficient with iturins. In this group, the peptidic diversity strictly depends on the selected strain. So far the screening remained the only method to discover new iturins. Another interesting peculiarity is the common occurrence in a single strain of two lipopeptides with different core structures such as surfactins and iturins. Taken together, these features led to an extensive metabolite pattern. Besides, engineered variants and chemical derivatives enlarged the array of available molecules. Despite the high degree of chemical similarity, the separation of variants and/or homologues was successfully achieved by reversed-phase HPLC leading to well-separated compounds ideally suited to investigation of structure-activity relationships. Improved physical techniques such as 2D-NMR and mass spectrometry allowed to describe efficiently and rapidly the composition of cyclic lipopeptides even in mixtures containing several variants. From NMR, the 3D structure and dynamics gave crucial data for fine structure-activity relationships as well as for understanding of the properties at the membrane and/or at the air/water interface. Here the role of residues was identified in the context of hydrophobic and electrostatic interactions that play a leader role. Such a comprehensive approach, based on both structural and biosynthesis knowledge, opened the way to rational design for enhanced properties and its validity was confirmed with 10 fold higher surfactant efficacy.

Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.

SummaryA 500 MHz 2D1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities,3JNH-αH coupling constants as well as1H/2H exchange rates and Δδ/ΔT temperature coefficients of NH protons strongly support the existence of an α-helix (residues 14–24) and of an antiparallel β-sheet (residues 27–40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from3JNH-αH coupling constants, (iii) 12 hydrogen bonds mostly deduced from1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a β-sheet is linked to an α-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Worldwide integrated assessment on systemic pesticides

In July 2009, a group of entomologists and ornithologists met at Notre Dame de Londres, a small village in the French department of Herault, as a result of an international enquiry amongst entomologists on the catastrophic decline of insects (and arthropods in general) all over Europe. They noted that a perceptible and gradual decline of insects, as part of the general impoverishment of the natural environment, had set in from the 1950s onwards. Amongstmany others, they recognized as root causes of this decline the intensification of agriculture with its accompanying loss of natural habitats and massive use of pesticides and herbicides, the manifold increase in roads and motorized traffic as well as a continent-wide nocturnal light pollution and nitrogen deposition. They equally agreed that a further degradation of the situation, a steeper decline in insect populations, had started in the decade 1990–2000. This first began inwestern Europe, followed by eastern and southern Europe, is nowadays apparent in the scarcity of insects splattered on windscreens of motorcars and squashed against their radiators and is best documented in the decline of butterflies and the global disorders amongst honey bees. They concluded that these phenomena reflected the now general collapse of Europe’s entomofauna. They also noted that the massive collapse of different species, genera and families of arthropods coincided with the severe decline of populations of different insectivorous bird species up to now considered as “common” such as swallows and starlings. On the basis of existing studies and numerous observations in the field as well as overwhelming circumstantial evidence, they came to the hypothesis that the new generation of pesticides, the persistent, systemic and neurotoxic neonicotinoids and fipronil, introduced in the early 1990s, are likely to be responsible at least in part for these declines. They, therefore, issued the Appeal of Notre Dame de Londres under the heading “No Silent Spring again” referring to Rachel Carson’s book “Silent Spring” then published almost half a century ago:

Production, isolation and characterization of (Leu4) - and (Ile4)surfactins from Bacillus subtilis

Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the l-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu4]- and [Ile4]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.

Androctonin, a novel antimicrobial peptide from scorpion Androctonus australis: solution structure and molecular dynamics simulations in the presence of a lipid monolayer.

Androctonin is a highly cationic antimicrobial peptide from scorpion exhibiting a broad spectrum of activities against bacteria and fungi. It contains 25 amino acids including four cysteine residues forming two disulfide bridges. We report here on the determination of its solution structure by conventional two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling using distance geometry and molecular dynamics methods. The structure of androctonin involves a well-defined highly twisted anti-parallel beta-sheet with strands connected by a more variable positively charged turn. A comparison with the structure of tachyplesin I (horseshoe crab) reveals that the amphiphilic character of the protein surface of this homologous peptide is not observed in androctonin. We have undertaken a 200-ps molecular dynamics simulation study on a system including one androctonin molecule and a monolayer of DMPG (1,2-dimyristoylphosphatidylglycerol) lipids. On the basis of this simulation, the first steps of the membrane permeabilization process are discussed.

Lipopeptides with Improved Properties: Structure by NMR, Purification by HPLC and Structure–Activity Relationships of New Isoleucyl‐rich Surfactins

The biosynthesis of bacterial isoleucyl‐rich surfactins was controlled by supplementation of L‐isoleucine to the culture medium. Two new variants, the [Ile4,7]‐ and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse‐phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified to β‐hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two‐dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure–activity relationships were discussed in details on the basis of the three‐dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu‐to‐Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

Acyl chain length dependence in the stability of melittin-phosphatidylcholine complexes. A light scattering and 31P-NMR study

Light scattering and 31P-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C16:0 to C20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, Ri > or = 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 A with dipalmitoyl-PC (DPPC) to 1500 A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 A radius when the system is cooled down below Tc (the gel-to-fluid phase transition temperature). Little chain length dependence is observed for the small objects. Small structures are also detected independently of the physical state of lipids (gel or fluid) when Ri < or = 5 and provided the interaction has been made above Tc. Small discs clearly characterized for DPPC and distearoyl-PC (DSPC) lipids are much less stable with DAPC. However in the long term, all these small structures fuse into large lipid lamellae. Discs are thermodynamically unstable and kinetics of disappearance of the small lipid-toxin complexes increases as the chain length increases in the sense: DAPC >> DSPC > DPPC. Kinetics of fusion of the small discs into extended bilayers is described by a pseudo-first-order law involving a lag time after which fusion starts. Increasing the chain length decreases the lag time and increases the rate of fusion. Formation of both the large vesicles in the fluid phase and the small discs in the gel phase as well as their stability is discussed in terms of relative shapes and dynamics of both lipids and toxin.

Refined solution structure of the anti‐mammal and anti‐insect LqqIII scorpion toxin: Comparison with other scorpion toxins

The solution structure of the anti‐mammal and anti‐insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatuswas refined and compared with other long‐chain scorpion toxins. This structure, determined by 1H‐NMR and molecular modeling, involves an α‐helix (18–29) linked to a three‐stranded β‐sheet (2–6, 33–39, and 43–51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 Å for Cα atoms. Comparison between LqqIII, the potent anti‐mammal AaHII, and the weakly active variant‐3 toxins revealed that the LqqIII three‐dimensional structure is closer to that of AaHII than to the variant‐3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long‐chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CSαβ (Cysteine Stabilized α‐helix β‐sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding‐sites of Na+ channels displays differences in the subsites interacting with anti‐mammal or anti‐insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long‐chain scorpion toxins. Proteins 28:360–374, 1997