Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Heterochiral Ala/(αMe)Aze sequential oligopeptides: Synthesis and conformational study

α‐Amino acid residues with a ?,ψ constrained conformation are known to significantly bias the peptide backbone 3D structure. An intriguing member of this class of compounds is (αMe)Aze, characterized by an N^α‐alkylated four‐membered ring and C^α‐methylation. We have already reported that (S)‐(αMe)Aze, when followed by (S)‐Ala in the homochiral dipeptide sequential motif ‐(S)‐(αMe)Aze‐(S)‐Ala‐, tends to generate the unprecedented γ‐bend ribbon conformation, as formation of a regular, fully intramolecularly H‐bonded γ‐helix is precluded, due to the occurrence of a tertiary amide bond every two residues. In this work, we have expanded this study to the preparation and 3D structural analysis of the heterochiral (S)‐Ala/(R)‐(αMe)Aze sequential peptides from dimer to hexamer. Our conformational results show that members of this series may fold in type‐II β‐turns or in γ‐turns depending on the experimental conditions.     

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

Accurate geometries for “Mountain pass” regions of the Ramachandran plot using quantum chemical calculations

Unusual local arrangements of protein in Ramachandran space are not well represented by standard geometry tools used in either protein structure refinement using simple harmonic geometry restraints or in protein simulations using molecular mechanics force fields. In contrast, quantum chemical computations using small poly‐peptide molecular models can predict accurate geometries for any well‐defined backbone Ramachandran orientation. For conformations along transition regions—ϕ from −60 to 60°—a very good agreement with representative high‐resolution experimental X‐ray (≤1.5 Å) protein structures is obtained for both backbone C⁻¹‐N‐C_α angle and the nonbonded O⁻¹…C distance, while “standard geometry” leads to the “clashing” of O…C atoms and Amber FF99SB predicts distances too large by about 0.15 Å. These results confirm that quantum chemistry computations add valuable support for detailed analysis of local structural arrangements in proteins, providing improved or missing data for less understood high‐energy or unusual regions.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of substituents on conformational preferences of helix foldamers of γ‐dipeptides

Conformational preferences of 9‐ and 14‐helix foldamers have been studied for γ‐dipeptides of 2‐aminocyclohexylacetic acid (γAc₆a) residues such as Ac‐(γAc₆a)₂‐NHMe ( 1 ), Ac‐(C^α‐Et‐γAc₆a)₂‐NHMe ( 2 ), Ac‐(γAc₆a)₂‐NHBn ( 3 ), and Ac‐(C^α‐Et‐γAc₆a)₂‐NHBn ( 4 ) at the M06‐2X/cc‐pVTZ//M06‐2X/6‐31 + G(d) level of theory to explore the influence of substituents on their conformational preferences. In the gas phase, the 9‐helix foldamer H9 and 14‐helix foldamer H14‐z are found to be most preferred for dipeptides 2 and 4 , respectively, as for dipeptides 1 and 3 , which indicates no remarkable influence of the C^α‐ethyl substitution on conformational preferences. The benzyl substitution at the C‐terminal end lead H14‐z to be the most preferred conformer for dipeptides 3 and 4 , whereas it is H9 for dipeptides 1 and 2 , which can be ascribed to the favored C? H···π interactions between the cyclohexyl group of the first residue and the C‐terminal benzyl group. There are only marginal changes in backbone structures and the distances and angles of H‐bonds for all local minima by C^α‐ethyl and/or benzyl substitutions. Although vibrational frequencies and intensities of the dipeptide 4 calculated at both M06‐2X/6‐31 + G(d) and M05‐2X/6‐31 + G(d) levels of theory are consistent with observed results in the gas phase, H14‐z is predicted to be most preferred by ΔG only at the former level of theory. Hydration did not bring the significant changes in backbone structures of helix foldamers for both dipeptide 1 and 4 . It is expected that the different substitutions at the C‐terminal end lead to the different helix foldamers, which may increase the resistance of helical structures to proteolysis and provide the more surface to the helical structures suitable for molecular recognition. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1077–1087, 2014.     

Revisiting the Ramachandran plot: hard-sphere repulsion, electrostatics, and H-bonding in the alpha-helix

What determines the shape of the allowed regions in the Ramachandran plot? Although Ramachandran explained these regions in terms of 1–4 hard‐sphere repulsions, there are discrepancies with the data where, in particular, the α_R, α_L, and β‐strand regions are diagonal. The α_R‐region also varies along the α‐helix where it is constrained at the center and the amino terminus but diffuse at the carboxyl terminus. By analyzing a high‐resolution database of protein structures, we find that certain 1–4 hard‐sphere repulsions in the standard steric map of Ramachandran do not affect the statistical distributions. By ignoring these steric clashes (N···H_i+1 and O_i?1···C), we identify a revised set of steric clashes (C^β···O, O_i?1···N_i+1, C^β···N_i+1, O_i?1···C^β, and O_i?1···O) that produce a better match with the data. We also find that the strictly forbidden region in the Ramachandran plot is excluded by multiple steric clashes, whereas the outlier region is excluded by only one significant steric clash. However, steric clashes alone do not account for the diagonal regions. Using electrostatics to analyze the conformational dependence of specific interatomic interactions, we find that the diagonal shape of the α_R and α_L‐regions also depends on the optimization of the N···H_i+1 and O_i?1···C interactions, and the diagonal β‐strand region is due to the alignment of the CO and NH dipoles. Finally, we reproduce the variation of the Ramachandran plot along the α‐helix in a simple model that uses only H‐bonding constraints. This allows us to rationalize the difference between the amino terminus and the carboxyl terminus of the α‐helix in terms of backbone entropy.     

Enantiomeric assay of escitalopram S(+)‐enantiomer and its “in‐process impurities” using two different techniques

The enantiomeric purity of escitalopram oxalate ESC and its “in‐process impurities,” namely, ESC‐N‐oxide, ESC‐citadiol, and R(?)‐enantiomer were studied in drug substance and products using high‐performance liquid chromatography (HPLC)‐UV (Method I), synchronous fluorescence spectroscopy (SFS) (Method IIA), and first derivative SFS (Method IIB). Method I describes as an isocratic HPLC‐UV for the direct resolution and determination of enantiomeric purity of ESC and its “in‐process impurities.” The proposed method involved the use of α_l‐acid glycoprotein (AGP) chiral stationary phase. The regression plots revealed good linear relationships of concentration range of 0.25 to 100 and 0.25 to 10 μg mL^?1 for ESC and its impurities. The limits of detection and quantifications for ESC were 0.075 and 0.235 μg mL^?1, respectively. Method II involves the significant enhancement of the fluorescence intensities of ESC and its impurities through inclusion complexes formation with hydroxyl propyl‐β‐cyclodextrin as a chiral selector in Micliavain buffer. Method IIA describes SFS technique for assay of ESC at 225 nm in presence of its impurities: R(?)‐enantiomer, citadiol, and N‐oxide at ?λ of 100 nm. This method was extended to (Method IIB) to apply first derivative SFS for the simultaneous determination of ESC at 236 nm and its impurities: the R(?)‐enantiomer, citadiol, and N‐oxide at 308, 275, and 280 nm, respectively. Linearity ranges were found to be 0.01 to 1.0 μg mL^?1 for ESC and its impurities with lower detection and quantification limits of 0.033/0.011 and 0.038/0.013 μg mL^?1 for SFS and first derivative synchronous fluorescence spectra (FDSFS), respectively. The methods were used to investigate the enantiomeric purity of escitalopram.     

Rings and ribbons in protein structures: Characterization using helical parameters and Ramachandran plots for repeating dipeptides

Helical parameters displayed on a Ramachandran plot allow peptide structures with successive residues having identical main chain conformations to be studied. We investigate repeating dipeptide main chain conformations and present Ramachandran plots encompassing the range of possible structures. Repeating dipeptides fall into the categories: rings, ribbons, and helices. Partial rings occur in the form of “nests” and “catgrips”; many nests are bridged by an oxygen atom hydrogen bonding to the main chain NH groups of alternate residues, an interaction optimized by the ring structure of the nest. A novel recurring feature is identified that we name unpleated β, often situated at the ends of a β‐sheet strand. Some are partial rings causing the polypeptide to curve gently away from the sheet; some are straight. They lack β‐pleat and almost all incorporate a glycine. An example is the first glycine in the GxxxxGK motif of P‐loop proteins. Ribbons in repeating dipeptides can be either flat, as seen in repeated type II and type II′ β‐turns, or twisted, as in multiple type I and type I′ β‐turns. Hexa‐ and octa‐peptides in such twisted ribbons occur frequently in proteins, predominantly with type I β‐turns, and are the same as the “β‐bend ribbons” hitherto identified only in short peptides. One is seen in the GTPase‐activating protein for Rho in the active, but not the inactive, form of the enzyme. It forms a β‐bend ribbon, which incorporates the catalytic arginine, allowing its side chain guanidino group to approach the active site and enhance enzyme activity. Proteins 2014; 82:230–239. © 2013 Wiley Periodicals, Inc.     

Solvent effects on the conformational preferences of model peptoids. MP2 study

The influence of aqueous environment on the main‐chain conformation (ω₀, ?, and ψ dihedral angles) of two model peptoids: N‐acetyl‐N‐methylglycine N’‐methylamide (Ac‐N(Me)‐Gly‐NHMe) ( 1 ) and N‐acetyl‐N‐methylglycine N’,N’‐dimethylamide (Ac‐N(Me)‐Gly‐NMe₂) ( 2 ) was investigated by MP2/6‐311++G(d,p) method. The Ramachandran maps of both studied molecules with cis and trans configuration of the N‐terminal amide bond in the gas phase and in water environment were obtained and all energy minima localized. The polarizable continuum model was applied to estimate the solvation effect on conformation. Energy minima of the Ac‐N(Me)‐Gly‐NHMe and Ac‐N(Me)‐Gly‐NMe₂ have been analyzed in terms of the possible hydrogen bonds and C = O dipole attraction. To validate the theoretical results obtained, conformations of the similar structures gathered in the Cambridge Crystallographic Data Centre were analyzed. Obtained results indicate that aqueous environment in model peptoids 1 and 2 favors the conformation F (? and ψ = ?70º, 180º), and additionally significantly increases the percentage of structures with cis configuration of N‐terminal amide bond in studied compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Difference in the structures of alanine tri‐ and tetra‐peptides with antiparallel β‐sheet assessed by X‐ray diffraction,solid‐state NMR and chemical shift calculations by GIPAW

Alanine oligomers provide a key structure for silk fibers from spider and wild silkworms.We report on structural analysis of l ‐alanyl‐l ‐alanyl‐l ‐alanyl‐l ‐alanine (Ala)₄ with anti‐parallel (AP) β‐structures using X‐ray and solid‐state NMR. All of the Ala residues in the (Ala)₄ are in equivalent positions, whereas for alanine trimer (Ala)₃ there are two alternative locations in a unit cell as reported previously (Fawcett and Camerman, Acta Cryst., 1975, 31, 658–665). (Ala)₄ with AP β‐structure is more stable than AP‐(Ala)₃ due to formation of the stronger hydrogen bonds. The intermolecular structure of (Ala)₄ is also different from polyalanine fiber structure, indicating that the interchain arrangement of AP β‐structure changes with increasing alanine sequencelength. Furthermore the precise ¹H positions, which are usually inaccesible by X‐ray diffraction method, are determined by high resolution ¹H solid state NMR combined with the chemical shift calculations by the gauge‐including projector augmented wave method. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 13–20, 2014.     

Cytotoxic (9βH)‐Pimarane and (9βH)‐17‐Norpimarane Diterpenes from the Tuber of Icacina trichantha

Three (9βH)‐pimaranes, 1, 2 , and 3 , and two (9βH)‐17‐norpimaranes, 4 and 5 , belonging to a rare compound class in nature, were obtained from the tubers of Icacina trichantha for the first time. Compound 1 is a new natural product, and 2 – 5 have been previously reported. The structures were elucidated based on NMR and MS data, and optical rotation values. The absolute configurations of (9βH)‐pimaranes were unambiguously established based on X‐ray crystallographic analysis. Full NMR signal assignments for the known compounds 2, 4 , and 5 , which were not available in previous publications, are also reported. All five isolates displayed cytotoxic activities on MDA‐MB‐435 cells (IC₅₀ 0.66–6.44 μM ), while 2, 3 , and 4 also exhibited cytotoxicities on HT‐29 cells (IC₅₀ 3.00–4.94 μM ).     

Introduction of a new scheme for classifying β-turns in protein structures

Protein β-turn classification remains an area of ongoing development in structural biology research. While the commonly used nomenclature defining type I, type II and type IV β-turns was introduced in the 1970s and 1980s, refinements of β-turn type definitions have been introduced as recently as 2019 by Dunbrack, Jr and co-workers who expanded the number of β-turn types to 18 (Shapovalov et al, PLOS Computat. Biol., 15, e1006844, 2019). Based on their analysis of 13 030 turns from 1074 ultrahigh resolution (≤1.2 Å) protein structures, they used a new clustering algorithm to expand the definitions used to classify protein β-turns and introduced a new nomenclature system. We recently encountered a specific problem when classifying β-turns in crystal structures of pentapeptide repeat proteins (PRPs) determined in our lab that are largely composed of β-turns that often lie close to, but just outside of, canonical β-turn regions. To address this problem, we devised a new scheme that merges the Klyne-Prelog stereochemistry nomenclature and definitions with the Ramachandran plot. The resulting Klyne-Prelog-modified Ramachandran plot scheme defines 1296 distinct potential β-turn classifications that cover all possible protein β-turn space with a nomenclature that indicates the stereochemistry of i + 1 and i + 2 backbone dihedral angles. The utility of the new classification scheme was illustrated by re-classification of the β-turns in all known protein structures in the PRP superfamily and further assessed using a database of 16 657 high-resolution protein structures (≤1.5 Å) from which 522 776 β-turns were identified and classified.     

Optically Active α‐Phenylethylamine as Efficient Organocatalyst in the Solvent‐free Reactions Between 2,3‐Butanedione and Conjugated Nitroolefins

(R)‐(+) and (S)‐(?)‐1‐phenylethylamine have been shown to promote highly diastereoselective and complementary enantioselective formal [3 + 2]carbocyclization reactions between 2,3‐butanedione and conjugated nitroalkenes with formation of enantiomerically rich 2‐hydroxy‐3‐nitrocyclopentanone derivatives. The reactions were carried out both in solvent and under solvent‐free conditions. The absolute configurations of the products were assigned by X‐ray and circular dichroism spectra analyses. Chirality 24:1005–1012, 2012. © 2012 Wiley Periodicals, Inc.     

Structure of an early native‐like intermediate of β2‐microglobulin amyloidogenesis

To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.     

A variety of changes,including CRISPR/Cas9‐mediated deletions,in CENH3 lead to haploid induction on outcrossing

Creating true‐breeding lines is a critical step in plant breeding. Novel, completely homozygous true‐breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere‐specific histone 3 variant (CENH3), including chimeric proteins, expression of non‐native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild‐type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS‐inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9‐mediated in‐frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild‐type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non‐transgenic approaches to the generation of haploid inducers.     

Pyrazole amino acids: hydrogen bonding directed conformations of 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue

A series of model compounds containing 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue with N‐terminal amide/urethane and C‐terminal amide/hydrazide/ester groups were investigated by using NMR, Fourier transform infrared, and single‐crystal X‐ray diffraction methods, additionally supported by theoretical calculations. The studies demonstrate that the most preferred is the extended conformation with torsion angles ? and ψ close to ±180°. The studied 1H‐pyrazole with N‐terminal amide/urethane and C‐terminal amide/hydrazide groups solely adopts this energetically favored conformation confirming rigidity of that structural motif. However, when the C‐terminal ester group is present, the second conformation with torsion angles ? and ψ close to ±180° and 0°, respectively, is accessible. The conformational equilibrium is observed in NMR and Fourier transform infrared studies in solution in polar environment as well as in the crystal structures of other related compounds. The observed conformational preferences are clearly related to the presence of intramolecular interactions formed within the studied residue. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Are Bacteria the Major Producers of Extracellular Glycolytic Enzymes in Aquatic Environments?

In aquatic microbial ecology, it has been considered that most extracellular enzymes except phosphatases are of bacterial origin. We tested this paradigm by evaluating the relationship between bacterial cell number and the activity of three glycolytic enzymes from 17 fresh waters and also from a laboratory experiment. Our large sets of pooled data do not seem to support such a simple explanation, because we found only a weak correlation of bacterial number with activity of α‐glucosidase (r_s = 0.63), β‐glucosidase (r_s = 0.45), and β‐N‐acetylhexosaminidase (r_s = 0.44). We also tested relations of the enzymatic activities to potential sources of natural substrates: dissolved organic carbon (DOC) and phytoplankton (as chlorophyll a). Their correlations with the enzymatic activities tested were very weak or insignificant. On the other hand, we found evidence for distinct producers of extracellular enzymes by analysing enzyme kinetics. The kinetics usually did not follow the simple Michaelis‐Menten model but a more complex one, indicating a mixture of two enzymes with distinct affinity to a substrate. In combination with size fractionation, we could sometimes even distinguish three or more different enzymes. During diatom blooms, the diatom biomass tightly correlated with β‐N‐acetylhexosaminidase activity (>4 μm fraction). We also documented very tight relationships between activity of both glucosidases and dry weight of Daphnia longispina (r_s = 1.0 and 0.60 for α‐ and β‐glucosidases, respectively) in an alpine clear‐water lake. Our data and evidence from other studies indicate that extracellular glycosidic activities in aquatic ecosystems cannot generally be assigned only to bacteria. Also invertebrate animals and other eukaryotes (fungi, diatoms, protozoa etc.) should be considered as potentially very important enzyme producers. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.