scholarly journals Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases

2021 ◽  
Vol 22 (13) ◽  
pp. 7136
Author(s):  
Piotr Henryk Małecki ◽  
Paweł Mitkowski ◽  
Elżbieta Jagielska ◽  
Karolina Trochimiak ◽  
Stéphane Mesnage ◽  
...  
Keyword(s):  
Amino Acids ◽  
Enterococcus Faecalis ◽  
High Efficiency ◽  
Peptide Bond ◽  
Side Chains ◽  
Bacterial Cells ◽  
Short Side ◽  
Binding Cleft ◽  
Binding Groove ◽  
Low Ionic Strength

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.

scholarly journals Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish.

1984 ◽  
Vol 84 (3) ◽  
pp. 403-422 ◽  
Author(s):  
J Caprio ◽  
R P Byrd
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ictalurus Punctatus ◽  
Olfactory Receptor ◽  
Olfactory Receptors ◽  
Receptor Potential ◽  
Side Chains ◽  
Short Side ◽  
Neutral Amino Acids ◽  
Receptor Sites

Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also possibly cysteine. The specificities of these olfactory transduction processes in the catfish are similar to those for the biochemically determined receptor sites for amino acids in other species of fishes and to amino acid transport specificities in tissues of a variety of organisms.

scholarly journals Temperature-induced phase transitions for amino acids with linear side chains

2014 ◽  
Vol 70 (a1) ◽  
pp. C170-C170
Author(s):  
Carl Henrik Görbitz ◽  
Pavel Karen ◽  
Michal Dusek ◽  
Václav Petříček
Keyword(s):  
Amino Acids ◽  
Low Temperature ◽  
Side Chain ◽  
Side Chains ◽  
Ambient Conditions ◽  
Short Side ◽  
Space Groups ◽  
Cell Parameters ◽  
Commensurate Phase ◽  
Chain Conformations

Two polymorphs are known to exist under ambient conditions for a number of amino acids (three for glycine). While investigations at high pressure have revealed a number of additional polymorphs, temperature-induced changes are rare. Low-temperature structures with modified side-chain conformations were identified for L- and DL-cysteine. Furthermore, racemates with linear side chains, such as DL-methionine and the non-standard DL-aminobutyric acid (DL-Abu), DL-aminopentanoic acid (DL-norvaline, DL-Nva) and DL-aminohexanoic acid (DL-norleucine, DL-Nle), undergo major crystalline rearrangements on transitions between P21/c and C2/c space groups [1], some of them entropy driven (disordering). As for the corresponding enantio-pure amino acids, we recently described related P21 and I2 structures at 105 K for L-Abu, both with Z' = 4 [2]. A short side-chain C–C bond (1.426 Å) in the only available CSD entry for L-Nle (at 298 K) [3] lead us to suspect that disorder could have been overlooked in the original refinement. L-Nva has not been described previously. We now present single-crystal X-ray determinations between 105 and 405 K for L-Abu, L-Nva and L-Nle, showing phase behavior of unprecedented complexity. For L-Abu and L-Nva we find three different forms in this temperature interval, while four different phases were found for L-Nle. Its known C2 structure with Z' = 1 prevails between 200 and 390 K, and the side chain is indeed disordered 2:1 over two positions. Above 390 K disorder is extensive; the space group remains C2 but cell parameters change. Upon cooling new low-temperature forms are observed at 200 and 170 K. Both are modulated, but to a different extent: data collected at 100 K reveal an almost commensurate phase, while the 180 K phase is fully incommensurate. This is, to our knowledge, the first observation of modulated structures for an amino acid, and also the first observations of major crystalline rearrangements akin to those seen for the corresponding racemates.

Responses of Olfactory Forebrain Units to Amino Acids in the Channel Catfish

2007 ◽  
Vol 97 (3) ◽  
pp. 2490-2498 ◽  
Author(s):  
Alexander A. Nikonov ◽  
John Caprio
Keyword(s):  
Amino Acids ◽  
Channel Catfish ◽  
Side Chains ◽  
Single Neurons ◽  
Short Side ◽  
Biologically Relevant ◽  
Acidic Amino Acids ◽  
Group I ◽  
Group Ii ◽  
The Many

A paucity of information exists concerning the processing of odorant information by single neurons in any vertebrate above the level of the olfactory bulb (OB). In this report, odorant specificity to four types of L-α-amino acids (neutral with long side-chains, neutral with short side-chains, basic and acidic), known biologically relevant odorants for teleosts, was determined for 217 spontaneously active forebrain (FB) neurons in the channel catfish. Group I FB units were identified that were excited by only one of four types of amino acids; no Group I unit was encountered that was excited by an acidic amino acid. The Group I FB units exhibited similar preferences as described previously for OB neurons, suggesting that no major modifications of olfactory information for at least some of these units occurred between the OB and FB. Evidence, however, for the convergence of odor information between the OB and FB was suggested by Group II FB units that exhibited a broader excitatory molecular receptive range (EMRR) than those of previously recorded types of OB units or the Group I FB units. Group II FB units were excited by both neutral and basic amino acids and a few also by acidic amino acids, EMRRs not observed previously in OB units. Stimulus-induced inhibition, likely for contrast enhancement, was also often observed for the many of the FB units encountered. The observed EMRRs of the FB units presently identified and those of the OB units previously studied are consistent with the ability of catfish to behaviorally discriminate these compounds.

The Kinetics of Inhibition of the Action of Thrombin by the Fibrinopeptides Formed during the Clotting of Fibrinogen

1966 ◽  
Vol 16 (01/02) ◽  
pp. 277-295 ◽  
Author(s):  
A Silver ◽  
M Murray
Keyword(s):  
Amino Acids ◽  
Competitive Inhibition ◽  
Amorphous Material ◽  
Peptide Bond ◽  
Initial Reaction ◽  
Reaction Products ◽  
Coagulation Mechanism ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Burk Plot

SummaryVarious investigators have separated the coagulation products formed when fibrinogen is clotted with thrombin and identified fibrinopeptides A and B. Two other peaks are observed in the chromatogram of the products of coagulation, but these have mostly been dismissed by other workers. They have been identified by us as amino acids, smaller peptides and amorphous material (37). We have re-chromatographed these peaks and identified several amino acids. In a closed system of fibrinogen and thrombin, the only reaction products should be fibrin and peptide A and peptide B. This reasoning has come about because thrombin has been reported to be specific for the glycyl-arginyl peptide bond. It is suggested that thrombin also breaks other peptide linkages and the Peptide A and Peptide B are attacked by thrombin to yield proteolytic products. Thrombin is therefore probably not specific for the glycyl-arginyl bond but will react on other linkages as well.If the aforementioned is correct then the fibrinopeptides A and B would cause an inhibition with the coagulation mechanism itself. We have shown that an inhibition does occur. We suggest that there is an autoinhibition to the clotting mechanism that might be a control mechanism in the human body.The experiment was designed for coagulation to occur under controlled conditions of temperature and time. Purified reactants were used. We assembled an apparatus to record visually the speed of the initial reaction, the rate of the reaction, and the density of the final clot formed after a specific time.The figures we derived made available to us data whereby we could calculate and plot the information to show the mechanism and suggest that such an inhibition does exist and also further suggest that it might be competitive.In order to prove true competitive inhibition it is necessary to fulfill the criteria of the Lineweaver-Burk plot. This has been done. We have also satisfied other criteria of Dixon (29) and Bergman (31) that suggest true competitive inhibition.

scholarly journals GC-TOF/MS-based metabolomics analysis to investigate the changes driven by N-Acetylcysteine in the plant-pathogen Xanthomonas citri subsp. citri

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Simone Cristina Picchi ◽  
Mariana de Souza e Silva ◽  
Luiz Leonardo Saldanha ◽  
Henrique Ferreira ◽  
Marco Aurélio Takita ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Plant Pathogen ◽  
Model Organism ◽  
Citrus Canker ◽  
Bacterial Cells ◽  
Xanthomonas Citri ◽  
Canker Disease ◽  
Potential Use

AbstractN-Acetylcysteine (NAC) is an antioxidant, anti-adhesive, and antimicrobial compound. Even though there is much information regarding the role of NAC as an antioxidant and anti-adhesive agent, little is known about its antimicrobial activity. In order to assess its mode of action in bacterial cells, we investigated the metabolic responses triggered by NAC at neutral pH. As a model organism, we chose the Gram-negative plant pathogen Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker disease, due to the potential use of NAC as a sustainable molecule against phytopathogens dissemination in citrus cultivated areas. In presence of NAC, cell proliferation was affected after 4 h, but damages to the cell membrane were observed only after 24 h. Targeted metabolite profiling analysis using GC–MS/TOF unravelled that NAC seems to be metabolized by the cells affecting cysteine metabolism. Intriguingly, glutamine, a marker for nitrogen status, was not detected among the cells treated with NAC. The absence of glutamine was followed by a decrease in the levels of the majority of the proteinogenic amino acids, suggesting that the reduced availability of amino acids affect protein synthesis and consequently cell proliferation.

Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture

2001 ◽  
Vol 68 (1) ◽  
pp. 117-129 ◽  
Author(s):  
ABDELGHANI OUMER ◽  
PILAR GAYA ◽  
ESTRELLA FERNÁNDEZ-GARCÍA ◽  
RAÚL MARIACA ◽  
SONIA GARDE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Enterococcus Faecalis ◽  
Free Amino Acids ◽  
Free Amino ◽  
Leuconostoc Mesenteroides ◽  
Aminopeptidase Activity ◽  
Hydrophobic Peptides ◽  
Bacteriocin Producer ◽  
Total Free Amino Acids ◽  
Adjunct Culture

Hispánico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4[ratio ]1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0–1·0 g/kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0·11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6×107 cfu/g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1·0 g bacteriocin-producing culture/kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1·0 g bacteriocin-producing culture/kg were 1·80-fold and 2·17-fold those in control cheese of the same age. Inoculating milk with 1·0 g/kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.

Ester side chains engineered quinoxaline based D-A copolymers for high-efficiency all-polymer solar cells

2021 ◽  
pp. 132551
Author(s):  
Liang Zeng ◽  
Ruijie Ma ◽  
Zhongxin Zhou ◽  
Tao Liu ◽  
Yiqun Xiao ◽  
...  
Keyword(s):  
Solar Cells ◽  
High Efficiency ◽  
Polymer Solar Cells ◽  
Side Chains

scholarly journals Isolation, purification and structure of exochelin MS, the extracellular siderophore from Mycobacterium smegmatis

1995 ◽  
Vol 305 (1) ◽  
pp. 187-196 ◽  
Author(s):  
G J Sharman ◽  
D H Williams ◽  
D F Ewing ◽  
C Ratledge
Keyword(s):  
Amino Acids ◽  
Mycobacterium Smegmatis ◽  
Methyl Esters ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Gallium Complex

The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to > 98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9.5), has a lambda max at 420 nm and a probable Ks for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY and TOCSY) 1H n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl) -beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.

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