Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post‐translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C‐terminal “follower” sequence, rather than the canonical N‐terminal “leader” sequence. We report herein the structural and biochemical characterization of BotP, a leucyl‐aminopeptidase‐like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N‐terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn²⁺ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro‐ mycin.     

Probing the Outstanding Local Hydrophobicity Increases in Peptide Sequences Induced by Incorporation of Trifluoromethylated Amino Acids

In order to achieve accurate determination of the local hydrophobicity increases in peptide sequences produced by incorporation of trifluoromethylated amino acids (TfmAAs), the chromatographic hydrophobicity indexes (?₀) of three series of tripeptides containing three unnatural trifluoromethylated amino acids have been measured and compared with those of their non‐fluorinated analogues. The hydrophobic contribution of each fluorinated amino acid was quantified by varying the position and the protection of (R)‐ and (S)‐α‐trifluoromethylalanine (TfmAla), (R)‐trifluoromethylcysteine (TfmCys), and (S)‐trifluoromethionine (TFM) in a short peptide sequence. As a general trend, strong increases in hydrophobicity were precisely measured, even exceeding the high hydrophobic contribution of the natural amino acid isoleucine. This study validates the incorporation of trifluoromethylated amino acids into peptide sequences as a rational strategy for the fine‐tuning of hydrophobic peptide–protein interactions.     

When the Leader Gets Loose: In Vivo Biosynthesis of a Leaderless Prenisin Is Stimulated by a trans‐Acting Leader Peptide

The nisin leader is believed to be crucial for nisin biosynthesis. Here, by using a construct completely lacking the leader peptide, we show that an up to fivefold‐dehydrated leaderless prenisin can be obtained, as judged by MALDI‐TOF MS, and that some of these species are biologically active, thus suggesting that at least three lanthionine rings are present. Notably, by expressing the leader peptide in trans together with the leaderless prenisin, we were able to increase the dehydration/cyclization efficiency of both NisB and NisC, but still with limited efficiency until the fifth dehydratable residue (Thr13) was processed, thereby enabling three rings to form. This, for the first time, demonstrates that 1) the leader is not absolutely necessary for the dehydration reaction of class I lantibiotics to occur in vivo; 2) the leader acts in trans in vivo; 3) the leader increases the efficiency of modification. Based on previous work and our current study, a model for the interactions of NisB and NisC with prenisin is proposed, in which the leader induces a more active conformation and/or productive complex formation of the biosynthetic machinery, and, when covalently bound, is involved in increasing the efficiency of dehydration to the C‐terminal end of the prenisin substrate molecule.     

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

A model is presented for the 3-D structure of the catalyticdomain of the putative leader peptidase NisP of Lactococcuslactis, and the interaction with its specific substrate, theprecursor of the lantibiotic nisin. This homology model is basedon the crystal structures of subtilisin BPN' and thermitasein complex with the inhibitor eglin. Predictions are made ofthe general protein fold, inserted loops, Ca²⁺ binding sites,aromatic interactions and electrostatic interactions of NisP.Cleavage of the leader peptide from precursor nisin by NisPis the last step in maturation of nisin. A detailed predictionof the substrate binding site attempts to explain the basisof specificity of NisP for precursor nisin. Specific acidicresidues in the SI subsite of the substrate binding region ofNisP appear to be of particular importance for electrostaticinteraction with the PI Arg residue of precursor nisin afterwhich cleavage occurs. The hydrophobic S4 subsite of NisP mayalso contribute to substrate binding as it does in subtilisins.Predictions of enzyme-substrate interaction were tested by proteinengineering of precursor nisin and determining susceptibilityof mutant precursors to cleavage by NisP. An unusual propertyof NisP predicted from this catalytic domain model is a surfacepatch near the substrate binding region which is extremely richin aromatic residues. It may be involved in binding to the cellmembrane or to hydrophobic membrane proteins, or it may serveas the recognition and binding region for the modified, hydrophobicC-terminal segment of precursor nisin. Similar predictions forthe tertiary structure and substrate binding are made for thehighly homologous protein EpiP, the putative leader peptidasefor the lantibiotic epidermin from Staphylococcus epidermidis,but EpiP lacks the aromatic patch. Based on these models, proteinengineering can be employed not only to test the predicted enzyme-substrateinteractions, but also to design lantibiotic leader peptidaseswith a desired specificity.     

A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto

Bacillus subtilis subsp. natto secretes the ComX_natto pheromone as a quorum‐sensing pheromone to produce poly‐γ‐glutamate for biofilm formation. The amino‐acid sequence of the pheromone is Lys‐Trp‐Pro‐Pro‐Ile‐Glu, and the tryptophan residue is post‐translationally modified with a farnesyl group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C‐terminal end of the precursor peptide ComX_natto. Here, we report the functional analysis of ComQ_natto, which catalyzes a unique farnesyl‐transfer reaction. ComQ_natto recognizes not only full‐length ComX_natto but also N‐ and/or C‐terminal truncated ComX_natto analogues and even a single tryptophan for modification with a farnesyl group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQ_natto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.     

Peptide Architecture: Adding an α‐Helix to the PYY Lysine Side Chain Provides Nanomolar Binding and Body‐Weight‐Lowering Effects

The gut hormone PYY3‐36 influences food intake and body weight via interaction with hypothalamic presynaptic Y2 receptors (Y2R). Novel Y2R‐selective analogues of PYY3‐36 are therefore potential drug candidates for the treatment of obesity. It has been hypothesized that PYY3‐36 and possibly also the related PP‐fold peptides, NPY and PP, bind to the membrane via their amphipathic α‐helix prior to receptor interaction. The PYY3‐36 amphipathic α‐helix causes the peptide to associate with the membrane, making it essential for Y receptor potency as it potentially guides the C‐terminal pentapeptide into the correct conformation for receptor activation. Based on this hypothesis, the importance of the amphipathic nature of PYY3‐36, as well as the ability of amphipathic α‐helices to interact in solution to form di‐ and tetramers, we redesigned the peptide architecture by addition of an amphipathic α‐helix via the Lys 4 side chain of PYY3‐36. Two different amphipathic sequences were introduced; first, PYY17‐31, the native α‐helix of PYY, and secondly, its retro counterpart, PYY31‐17, which is also predicted to form an α‐helix. Moreover, several different turn motifs between the branching point and the additional α‐helix were tested. Several novel peptides with nanomolar Y2R binding affinities, as well as increased Y receptor selectivity, were identified. CD experiments showed the modifications to be well accepted, and an increase in mean ellipticity (ME) signifying an increased degree of α‐helicity was observed. Receptor binding experiments indicated that the direction of the additional α‐helix is less important, in contrast to the turn motifs, which greatly affect the Y1R binding and thus determine the Y1R activity. Conversely, the structure–activity relationships from in vivo data showed that the peptide containing the retro‐sequence was inactive, even though the binding data demonstrated high affinity and selectivity. This demonstrates that radical redesign of peptide architecture can provide nanomolar binding with improved subtype selectivity and with in vivo efficacy.     

Ribosomal Synthesis of Natural‐Product‐Like Bicyclic Peptides in Escherichia coli

Methods to access natural‐product‐like macrocyclic peptides can disclose new opportunities for the exploration of this important structural class for chemical biology and drug discovery applications. Here, the scope and mechanism of a novel strategy for directing the biosynthesis of thioether‐bridged bicyclic peptides in bacterial cells was investigated. This method entails split intein‐catalyzed head‐to‐tail cyclization of a ribosomally produced precursor peptide, combined with inter‐side‐chain crosslinking through a genetically encoded cysteine‐reactive amino acid. This strategy could be successfully applied to achieve formation of structurally diverse bicyclic peptides with high efficiency and selectivity in Escherichia coli. Insights into the sequence of reactions underlying the peptide bicyclization process were gained from time‐course experiments. Finally, the potential utility of this methodology toward the discovery of macrocyclic peptides with enhanced functional properties was demonstrated through the isolation of a bicyclic peptide with sub‐micromolar affinity for streptavidin.     

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers

Trichogin GA IV is a short peptaibol with antimicrobial activity. This uncharged, but amphipathic, sequence is aligned at the membrane interface and undergoes a transition to an aggregated state that inserts more deeply into the membrane, an assembly that predominates at a peptide-to-lipid ratio (P/L) of 1:20. In this work, the natural trichogin sequence was prepared and reconstituted into oriented lipid bilayers. The ¹⁵N NMR chemical shift is indicative of a well-defined alignment of the peptide parallel to the membrane surface at P/Ls of 1:120 and 1:20. When the P/L is increased to 1:8, an additional peptide topology is observed that is indicative of a heterogeneous orientation, with helix alignments ranging from around the magic angle to perfectly in-plane. The topological preference of the trichogin helix for an orientation parallel to the membrane surface was confirmed by attenuated total reflection FTIR spectroscopy. Furthermore, ¹⁹F CODEX experiments were performed on a trichogin sequence with ¹⁹F-Phe at position 10. The CODEX decay is in agreement with a tetrameric complex, in which the ¹⁹F sites are about 9–9.5 Å apart. Thus, a model emerges in which the monomeric peptide aligns along the membrane surface. When the peptide concentration increases, first dimeric and then tetrameric assemblies form, made up from helices oriented predominantly parallel to the membrane surface. The formation of these aggregates correlates with the release of vesicle contents including relatively large molecules.     

Biosynthetic Gene Cluster of a d‐Tryptophan‐Containing Lasso Peptide,MS‐271

MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271.     

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post‐translational modification of the precursor McjA by the ATP‐dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three‐dimensional folding of MccJ25. We further showed that the N‐terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43–S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23–Q34 in the N‐terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

Influence of Lipid Saturation,Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵LAL⁸LALALAL¹⁶ALW¹⁹LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ²H-labeled alanine residues within the central sequence for detection by solid-state ²H NMR spectroscopy. The resulting pattern of [²H]Ala quadrupolar splitting (Δν_q) magnitudes indicates the core helix for R^8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R^8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.     

Fabrication of durable hydrophobic cellulose surface from silane‐functionalized silica hydrosol via electrochemically assisted deposition

Durable excellent hydrophobic surface on cellulose substrate was fabricated from the silica hydrosol functionalized with silane chemicals by a facile electrochemically assisted deposition technique. The silica hydrosol was synthesized using tetraethoxysilane (TEOS) as the precursor and sodium dodecylbenzene (SDBS) as the emulsifier under acidic conditions. The hydrophobic silane modifiers including octyltriethoxysiliane (OTES), dodecyltriethoxysiliane (DTES) and isooctyltriethoxysiliane (iso‐OTES) and the silane‐coupling agent γ‐mercaptopropyltriethoxysilane (MPTES) were used to dope the silica hydrosol for preparing durable hydrophobic cellulose surface. The cellulose surface modified with silane modifier iso‐OTES exhibited the best hydrophobicity with water contact angle of 162.3 ± 0.5° due to its non‐polar and hydrolytically stable of ? Si(C₈H₁₇) groups. The addition of silane‐coupling agent MPTES containing the ? SH group led to good durability of hydrophobicity with water contact angle of 130.0 ± 1.2° after 20 washing times. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42733.     

Structural and Functional Analysis of Human Liver‐Expressed Antimicrobial Peptide 2

Human liver‐expressed antimicrobial peptide 2 (LEAP‐2) is a cationic antimicrobial peptide (CAMP) believed to have a protective role against bacterial infection. Little is known about the structure–activity relationships of LEAP‐2 or its mechanism of action. In this study we describe the structure of LEAP‐2, analyze its interaction with model membranes, and relate them to the antimicrobial activity of the peptide. The structure of LEAP‐2, determined by NMR spectroscopy, reveals a compact central core with disorder at the N and C termini. The core comprises a β‐hairpin and a 3₁₀‐ helix that are braced by disulfide bonds between Cys17–28 and Cys23–33 and further stabilized by a network of hydrogen bonds. Membrane‐affinity studies show that LEAP‐2 membrane binding is governed by electrostatic attractions, which are sensitive to ionic strength. Truncation studies show that the C‐terminal region of LEAP‐2 is irrelevant for membrane binding, whereas the N‐terminal (hydrophobic domain) and core regions (cationic domain) are essential. Bacterial‐growth‐inhibition assays reveal that the antimicrobial activity of LEAP‐2 correlates with membrane affinity. Interestingly, the native and reduced forms of LEAP‐2 have similar membrane affinity and antimicrobial activities; this suggests that disulfide bonds are not essential for the bactericidal activity. This study reveals that LEAP‐2 has a novel fold for a CAMP and suggests that although LEAP‐2 exhibits antimicrobial activity under low‐salt conditions, there is likely to be another physiological role for the peptide.     

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

Structural Basis for Antimicrobial Activity of Lasiocepsin

Lasiocepsin is a unique 27‐residue antimicrobial peptide, isolated from Lasioglossum laticeps (wild bee) venom, with substantial antibacterial and antifungal activity. It adopts a welldefined structure consisting of two α‐helices linked by a structured loop. Its basic residues form two distinct positively charged regions on the surface whereas aliphatic side chains contribute to solvent‐accessible hydrophobic areas, thus emphasising the amphipathic character of the molecule. Lasiocepsin structurally belongs to the ShK family and shows a strong preference for anionic phospholipids; this is further augmented by increasing concentrations of cardiolipin, such as those found at the poles of bacterial cells. The membrane‐permeabilising activity of the peptide is not limited to outer membranes of Gram‐negative bacteria. The peptide interacts with phospholipids initially through its N terminus, and its degree of penetration is strongly dependent on the presence of cardiolipin.     

Macrocyclization of Organo‐Peptide Hybrids through a Dual Bio‐orthogonal Ligation: Insights from Structure–Reactivity Studies

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo‐biosynthetic strategy for generating macrocyclic organo‐peptide hybrids (MOrPHs) through a dual oxime‐/intein‐mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3‐amino‐thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2‐amino‐ mercaptomethyl‐aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the “i?1” site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side‐chain→C‐end ligation versus C‐end→side‐chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo‐peptide macrocycles and provide valuable structure–reactivity insights to inform the construction of macrocycle libraries through this chemo‐biosynthetic strategy.     

Design of a Short Thermally Stable α‐Helix Embedded in a Macrocycle

Although helices play key roles in peptide–protein and protein–protein interactions, the helical conformation is generally unstable for short peptides (10–15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain‐to‐side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non‐hydrogen‐bonded amide groups to solvent. Here, we develop a “capped‐strapped” peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end‐capping interactions. We have designed a ten‐residue capped‐strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T_m≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X‐ray crystallography revealed a novel quaternary structure with implications for foldamer design.     

Crystal Structure of the Human Monoacylglycerol Lipase,a Key Actor in Endocannabinoid Signaling

2‐Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2‐arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N‐arachidonylmaleimide, a sulfhydryl‐reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2‐arachidonoylglycerol transmission.     

In vitro synthesis of polyhydroxyalkanoate catalyzed by class II and III PHA synthases: a useful technique for surface coatings of a hydrophobic support with PHA

In vitro synthesis of polyhydroxyalkanoates (PHAs) on a hydrophobic support, i.e. highly oriented pyrolytic graphite (HOPG), was performed using class II PHA synthase (PhaC1_Pp) from Pseudomonas putida and class III PHA synthase (PhaEC_Av) from Allochromatium vinosum. Using PhaC1_Pp and 3‐hydroxyoctanoyl‐CoA, a poly(3‐hydroxyoctanoate) [P(3HO)] film was formed on the hydrophobic support with a thickness of a few nanometers, as revealed by atomic force microscopy (AFM). A poly(3‐hydroxybutyrate) [P(3HB)] film was also formed using PhaEC_Av and 3‐hydroxybutyryl‐CoA. AFM observations of the HOPG surface during P(3HB) film formation revealed that polymerized P(3HB) covered HOPG surface within the reaction time, resulting in the formation of a homogeneous ultra‐thin film. This method of ultra‐thin PHA film formation on a hydrophobic support may be applicable to the surface‐coating technique of materials with biodegradable, bioabsorbable, and biocompatible PHAs. Copyright © 2009 Society of Chemical Industry