Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Bacterial β‐Aminopeptidases: Structural Insights and Applications for Biocatalysis

β‐Aminopeptidases comprise a class of enzymes with functional and structural similarities. All members of the β‐aminopeptidases described to date were isolated from bacterial sources. Uniquely, they catalyze the hydrolysis of β³‐ and/or β²‐amino acid residues from amides and peptides that are otherwise considered proteolytically stable. Due to this unusual reactivity with β‐peptide substrates, β‐aminopeptidases have potential to be used as biocatalysts for β‐peptide synthesis and for the resolution of enantiomerically pure β‐amino acids from racemic substrate mixtures. β‐Aminopeptidases are formed from an inactive precursor by posttranslational autoproteolytic cleavage, exposing the catalytic nucleophile at the N‐terminus of the newly formed β‐polypeptide chain. Such an activation step is a characteristic trait of enzymes of the N‐terminal nucleophile (Ntn) hydrolase superfamily. However, classical Ntn hydrolases and β‐aminopeptidases differ by the fold of their catalytic cores and are hence likely to originate from distinct evolutionary ancestors. In this contribution, we review the existing literature on β‐aminopeptidases, including biochemical and functional studies, as well as structural investigations that recently allowed insights into the catalytic mechanisms of precursor processing and β‐peptide conversion.     

How calcium inhibits the magnesium‐dependent kinase gsk3β: A molecular simulation study

Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine kinase that plays a pivotal role in many biological processes. GSK3β catalyzes the transfer of γ‐phosphate of ATP to the unique substrate Ser/Thr residues with the assistance of two natural activating cofactors Mg²⁺. Interestingly, the biological observation reveals that a non‐native Ca²⁺ ion can inhibit the GSK3β catalytic activity. Here, the inhibitory mechanism of GSK3β by the displacement of native Mg²⁺ at site 1 by Ca²⁺ was investigated by means of 80 ns comparative molecular dynamics (MD) simulations of the GSK3β···Mg²⁺‐2/ATP/ Mg²⁺‐1 and GSK3β···Mg²⁺‐2/ATP/Ca²⁺‐1 systems. MD simulation results revealed that using the AMBER point charge model force field for Mg²⁺ was more appropriate in the reproduction of the active site architectural characteristics of GSK3β than using the magnesium‐cationic dummy atom model force field. Compared with the native Mg²⁺ bound system, the misalignment of the critical triphosphate moiety of ATP, the erroneous coordination environments around the Mg²⁺ ion at site 2, and the rupture of the key hydrogen bond between the invariant Lys85 and the ATP O^β2 atom in the Ca²⁺ substituted system were observed in the MD simulation due to the Ca²⁺ ion in active site in order to achieve its preferred sevenfold coordination geometry, which adequately abolish the enzymatic activity. The obtained results are valuable in understanding the possible mechanism by why Ca²⁺ inhibits the GSK3β activity and also provide insights into the mechanism of Ca²⁺ inhibition in other structurally related protein kinases. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Bos indicus introgression into (peri‐)alpine cattle breeds – evidence from the analysis of bovine whey protein variants

The major bovine whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG), exhibit breed‐specific genetic variation. The aim of this study was to identify possible new protein variants and determine the distribution of variants across a variety of 18 taurine and indicine cattle breeds applying a DNA‐based sequencing approach. To this end, the open reading frames of the respective genes (LALBA and LGB) were sequenced in 476 animals. Within the LALBA gene, a previously unknown synonymous and a previously undesignated non‐synonymous nucleotide exchange were identified. Furthermore, two known α‐LA variants (A and B) and four known β‐LG variants (A, B, C and W) were determined. The occurrence of typical indicine variants in some taurine cattle breeds, such as Suisse Eringer, German Hinterwälder and Hungarian Grey Steppe, further supports the hypothesis of ancient Bos indicus introgression into (peri‐)alpine cattle breeds.     

Autophosphorylation of carboxy‐terminal residues inhibits the activity of protein kinase CK1α

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1δ and CK1ε were previously shown to autophosphorylate carboxy‐terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1α and splice variant CK1αL has identified the autophosphorylation of the last four carboxyl‐end serines and threonines and also for CK1αS, the same four residues plus threonine‐327 and serine‐332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy‐terminal phosphorylation sites of CK1α to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1α and CK1αS with λ phosphatase causes an activation of 80–100% and 300%, respectively. Similar treatment fails to stimulate the CK1α mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1α and CK1αS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1α and CK1αS was also observed using the known CK1 protein substrates DARPP‐32, β‐catenin, and CK2β, which have different CK1 recognition sequences. Autophosphorylation effects on CK1α activity are not due to changes in Km_app for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1α and its splice variants can be regulated by their autophosphorylation status. J. Cell. Biochem. 106: 399–408, 2009. © 2008 Wiley‐Liss, Inc.     

Integrated Genome and Protein Editing Swaps α‐2,6 Sialylation for α‐2,3 Sialic Acid on Recombinant Antibodies from CHO

Immunoglobin G with α‐2,6 sialylation has been reported to have an impact on antibody‐dependent cellular cytotoxicity and anti‐inflammatory efficacy. However, production of antibodies with α‐2,6 sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N‐glycan site and the presence of exclusively α‐2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α‐2,6 sialyltransferase produced IgG with significant levels of both α‐2,6 and α‐2,3 sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α‐2,3 sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α‐2,6 sialylation level relative to α‐2,3 sialylation for the α‐2,3 sialyltransferase knockouts when combined with α‐2,6 sialyltransferase overexpression. Indeed, α‐2,3 linked sialic acids were not detected on IgG produced from the α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α‐2,3 sialyltransferase knockout‐α‐2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI‐TOF and LC‐ESI‐MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N‐glycans with different structural variations for examining the role of glycosylation on protein performance.     

Studies of Amyloid‐Like Fibrillogenesis through β‐Sheet‐Mediated Self‐Assembly of Short Synthetic Peptides

Three structurally different types of small peptides, namely, i) Boc‐Ile‐Aib‐Ile‐OMe (Aib=α‐aminoisobutyric acid), ii) Boc‐Xx‐m‐aminobenzoic acid (Xx=β‐Ala and γ‐aminobutyric acid), and iii) Boc‐Xx‐m‐nitroaniline, were found to exhibit β‐sheet‐mediated fibrillogenesis in the solid state, revealed by FT‐IR, single‐crystal X‐ray diffraction, and FE‐SEM studies. Interestingly, the fibrils grown from peptides 2 and 3 were found to bind with the physiological dye Congo red, a characteristic feature of amyloid fibrils.     

Synthesis of β‐Ketoamide Curcumin Analogs for Anti‐Diabetic and AGEs Inhibitory Activities

Two different series of novel β‐ketoamide curcumin analogs enriched in biological activities have been synthesized. The synthesized compounds were screened for their in vitro anti‐diabetic and AGEs inhibitory activities and exhibited potent to good anti‐diabetic and AGEs inhibitory activities. The molecular docking study was also performed with the α‐amylase enzyme.     

On 3LEZ,a Deep‐Sea Halophilic Protein with in vitro Class‐A β‐Lactamase Activity: Molecular‐Dynamics,Docking, and Reactivity Simulations

This work shows that a deep‐sea protein, 3LEZ, with known in vitro β‐lactamase activity, proved stable, substantially in the conformation detected by X‐ray diffraction of the crystal, when subjected to molecular‐dynamics (MD) simulations under conditions compatible with shallow seas. Docking simulations showed that the β‐lactamase active site S85 of 3LEZ (S70 in Ambler numbering) is the preferential binding pocket for not only β‐lactam antibiotics and inhibitors, but, surprisingly, also for a wide variety of other biologically active compounds in various chemical classes, including marine metabolites. In line with the in vitro β‐lactamase activity, a) affinities on docking β‐lactam antibiotics and inhibitors onto 3LEZ were found to roughly parallel published K_m and K_i values, obtained from Michaelis? Menten kinetics under room conditions, and b) DFT calculations agreed with experiments that the irreversible reaction of the β‐lactamase inhibitor clavulanic acid with the whole S85 catalytic center of 3LEZ is spontaneous. These observations must be viewed in the light that a) the compounds in other chemical classes showed comparable affinities, and, in some cases, even higher than β‐lactams, for the S85 active site, b) K_m and K_i data are not available at the high hydrostatic pressure of the deep sea, where 3LEZ is believed to have evolved, c) an inverse order of affinities for the β‐lactams, with respect to both experimentation and simulations at room conditions, was observed from comparative docking simulations with 3LEZ derived from MD under high hydrostatic pressure. Although MD requires a general assessment for high hydrostatic pressure before c) above is given the same weight as all other observations, this work questions the conclusion that the in vitro determined β‐lactamase activity represents the ecological role of 3LEZ.     

Synthesis,binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3‐homo‐amino acids

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β³‐homo‐amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β³‐homo‐Tyr in place of Tyr (Tyr‐d ‐Ala‐Phe‐Gly‐β³‐homo‐Tyr‐NH‐)₂ showed good μ receptor affinity and selectivity (IC₅₀ = 0.302, IC₅₀ ratio μ/δ = 68) and enzymatic stability in human plasma. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Chemotaxonomic Importance of the Essential‐Oil Composition in Two Subspecies of Teucrium stocksianum Boiss. from Iran

The hydrodistilled essential oils obtained from aerial flowering parts of Teucrium stocksianum ssp. stocksianum (TSS) and T. stocksianum ssp. gabrielae (TSG) from Iran were analyzed by capillary GC and GC/MS. The oil analysis of two subspecies led to the identification of 65 compounds that accounted for 93.3 and 95.1% of the total oil compositions, respectively. Sesquiterpenoids (52.9%) constituted the main compounds in the essential oil of TSS represented mainly by cis‐sesquisabinene hydrate (12.0%), followed by epi‐β‐bisabolol (6.6%), guaiol (5.4%), and β‐eudesmol (4.4%), whilst monoterpenoids (61.2%) were found to be the major components of the oil of TSG, represented by α‐pinene (23.0%), β‐pinene (13.0%), myrcene (6.3%), and sabinene (6.3%). The principal component in both subspecies, TSS and TSG, was α‐pinene (22.0 and 23.0%, resp.) and β‐pinene (6.5 and 13.0%, resp.). epi‐α‐Cadinol, myrcene, and sabinene, which were detected as principal compounds of TSG, were characterized in lower amounts (<1.5%) in the oil of TSS. Seven components were identified in the oil of TSS corresponding to 25.9% of total oil, which were totally absent in the oil of TSG, of which cis‐sesquisabinene hydrate (12.0%), guaiol (5.4%), and β‐eudesmol (4.4%) were in considerable amounts. Taxonomic position of the subspecies is discussed on the basis of phytochemical data.     

High tolerance to simultaneous active‐site mutations in TEM‐1 β‐lactamase: Distinct mutational paths provide more generalized β‐lactam recognition

The diversity in substrate recognition spectra exhibited by various β‐lactamases can result from one or a few mutations in the active‐site area. Using Escherichia coli TEM‐1 β‐lactamase as a template that efficiently hydrolyses penicillins, we performed site‐saturation mutagenesis simultaneously on two opposite faces of the active‐site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended‐spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (K_M) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (k_cat) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM‐1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.     

Modified level of miR‐376a is associated with Parkinson's disease

Parkinson's disease (PD) is a frequent progressive neurodegenerative disorder. Impaired mitochondrial function is a major feature of sporadic PD. Some susceptibility or causative genes detected in PD are strongly associated with mitochondrial dysfunction including PGC1α, TFAM and GSK3β. microRNAs (miRNAs) are non‐coding RNAs whose altered levels are proven in disparate PD models and human brains. Therefore, the aim of this study was to detect modulations of miRs upstream of PGC1α, TFAM and GSK3β in association with PD onset and progress. In this study, a total of 33 PD subjects and 25 healthy volunteers were recruited. Candidate miRNA (miR‐376a) was selected through target prediction tools and literature survey. Chronic and acute in vitro PD models were created by MPP⁺‐intoxicated SHSY5Y cells. The levels of miR‐376a and aforementioned genes were assessed by RT‐qPCR. The expression of target genes was decreased in chronic model while there were dramatically up‐regulated levels of those genes in acute model of PD. miR‐376a was strongly altered in both acute and chronic PD models as well as PBMCs of PD patients. Our results also showed overexpression of PGC1α, and TFAM in PBMCs is inversely correlated with down‐regulation of miR‐376a, suggesting that miR‐376a possibly has an impact on PD pathogenesis through regulation of these genes which are involved in mitochondrial function. miR‐376a expression in PD‐derived PBMCs was also correlated with disease severity and may serve as a potential biomarker for PD diagnosis. This is the first study showing altered levels of miR‐376a in PD models and PBMCs, suggesting the probable role of this miRNA in PD pathogenesis. The present study also proposed TFAM and PGC1α as target genes of miR‐376a for the first time, through which it possibly can exert its impact on PD pathogenesis.     

Progesterone 5β‐reductase genes of the Brassicaceae family as function‐associated molecular markers

This study aimed to define progesterone 5β‐reductases (P5βR, EC 1.3.99.6, enone 1,4‐reductases) as function‐associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His‐tagged forms of the P5βR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4‐reductase activity was demonstrated in vitro using progesterone and 2‐cyclohexen‐1‐one as substrates. Evidence is provided that functional P5βRs are ubiquitous in the Brassicaceae. The recombinant P5βR enzymes showed different substrate preferences towards progesterone and 2‐cyclohexen‐1‐one. Sequence comparison of the catalytic pocket of the P5βR enzymes and homology modelling using Digitalis lanata P5βR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5βR genes or P5βR proteins can be used as valuable function‐associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective.     

Inhibitory effects of indole α‐lipoic acid derivatives on nitric oxide production in LPS/IFNγ activated RAW 264.7 macrophages

Alpha‐lipoic acid (α‐lipoic acid) is a potent antioxidant compound that has been shown to possess anti‐inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL‐1β, IL‐6 and TNF‐alpha upon activation with LPS ( Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α‐lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α‐lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I‐4b, I‐4e and II‐3b. In conclusion, these indole α‐lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of solvent fractions of Allomyrina dichotoma larvae through the inhibition of in vitro BACE1 and β‐amyloid(25–35)‐induced toxicity in rat pheochromocytoma PC12 cells

Amyloid‐β peptide (Aβ) generation initiated by β‐site amyloid precursor protein cleaving enzyme 1 BACE1 is a critical cause of Alzheimer's disease. In the course of our ongoing investigation of natural anti‐dementia resources, the ethyl acetate (EtOAc) fraction exerted strong BACE1‐specific inhibition with the half maximal inhibitory concentration (IC₅₀) value of 9.2 × 10^?5 μg/mL. Furthermore, Aβ(25–35)‐induced cell death was predominantly prevented by the EtOAc fraction of Allomyrina dichotoma larvae through diminishing of cellular oxidative stress and attenuating apoptosis by inhibiting caspase‐3 activity. Taken together, the present study demonstrated that A. dichotoma larvae possess novel neuroprotective properties not only via the selective and specific inhibition of BACE1 activity but also through the alleviation of Aβ(25–35)‐induced toxicity, which may raise the possibility of therapeutic application of A. dichotoma larvae for preventing and/or treating dementia.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.