scholarly journals The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

2014 ◽  
Vol 80 (24) ◽  
pp. 7561-7573 ◽  
Author(s):  
Jean-Baptiste Fournier ◽  
Etienne Rebuffet ◽  
Ludovic Delage ◽  
Romain Grijol ◽  
Laurence Meslet-Cladière ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Content Type ◽  
Bacterial Phyla ◽  
Iodide Oxidation ◽  
Enzyme Family ◽  
Evolutionary Features ◽  
Eukaryotic Species ◽  
Monomeric Structure

ABSTRACTVanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacteriumZobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced inEscherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungusCurvularia inaequalisand ofStreptomycessp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.

scholarly journals Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase

2011 ◽  
Vol 77 (23) ◽  
pp. 8409-8414 ◽  
Author(s):  
Yong-Sheng Tian ◽  
Jing Xu ◽  
Ai-Sheng Xiong ◽  
Wei Zhao ◽  
Xiao-Yan Fu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Three Dimensional ◽  
Glyphosate Resistance ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Ochrobactrum Anthropi ◽  
Glyphosate Tolerance ◽  
Content Type ◽  
Important Region ◽  
Phosphate Synthase

ABSTRACTA mutant of 5-enopyruvylshikimate-3-phosphate synthase fromOchrobactrum anthropiwas identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenicArabidopsiswas obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative

2003 ◽  
Vol 89 (01) ◽  
pp. 74-82 ◽  
Author(s):  
Koen Verbeke ◽  
Ann Gils ◽  
Jean-Marie Stassen ◽  
Paul Declerck
Keyword(s):  
Rational Design ◽  
Three Dimensional ◽  
Neutralizing Antibody ◽  
Plasminogen Activator Inhibitor ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryInterfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (KA = 4.8 ± 0.2 × 107 M–1 vs. 1.8 ± 0.7 × 109 M–1 for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (KA = 2.1 ± 0.2 × 107 M–1).In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.

scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

Hospitality situations, consumer expertise, and perceptions of wine attributes: three empirical studies

2019 ◽  
Vol 31 (1) ◽  
pp. 68-88 ◽  
Author(s):  
Dale F. Duhan ◽  
Shannon B. Rinaldo ◽  
Natalia Velikova ◽  
Tim Dodd ◽  
Brent Trela
Keyword(s):  
Empirical Studies ◽  
Three Dimensional ◽  
Total Sample ◽  
Dimensional Structure ◽  
Product Knowledge ◽  
Product Attributes ◽  
Content Type ◽  
Wine Consumption ◽  
Consumer Expertise ◽  
Academic Researchers

PurposeWine choices are not always fully understood by academic researchers or the industry. This paper aims to outline and test a theoretical model proposing that wine consumption may be dependent on differences in consumer expertise, the hospitality situation, characteristics of the wine itself and an interaction of these variables.Design/methodology/approachThree empirical studies (total sample size = 356) tested these theoretical propositions. Consumers with varying levels of wine knowledge were presented with experimental vignettes showing videos of wine opening and pouring and were asked to pair wines with hospitality situations.FindingsStudy 1 found that consumers with low product knowledge were more sensitive to hospitality situations and extrinsic product attributes (closures) than were the experts. Study 2 found that wine hospitality situations fall into three predicted categories, namely, food, friends and formality, although contrary to prediction, the presence of food was the weakest predictors. Study 3 demonstrated the robustness of the three-dimensional structure of wine hospitality situations.Practical implicationsThese studies provided important practical information because targeting various market segments requires the industry to know what product attributes are favored by different groups of consumers different situations.Originality/valuePrevious researchers have discussed the difficulty of measuring consumption situations. By limiting these studies to wine consumption within hospitality situations, the authors learned much about how consumers’ characteristics, product attributes and the situations interact to influence not only product assessments but also choices.

scholarly journals Older workers: stereotypes and occupational self-efficacy

2016 ◽  
Vol 31 (7) ◽  
pp. 1152-1166 ◽  
Author(s):  
Rita Chiesa ◽  
Stefano Toderi ◽  
Paola Dordoni ◽  
Kene Henkens ◽  
Elena Maria Fiabane ◽  
...  
Keyword(s):  
Older Workers ◽  
Self Efficacy ◽  
Age Groups ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Age Stereotypes ◽  
Content Type ◽  
Moderator Role ◽  
The Relationship ◽  
Organizational Age

Purpose The purpose of this paper is to explore the relationship between organizational age stereotypes and occupational self-efficacy. First, the authors intend to test the measurement invariance of Henkens’s (2005) age stereotypes scale across two age group, respectively, under 50 and 50 years and older. Then, the moderator role of age groups in the relationship between age stereotypes and occupational self-efficacy is investigated. Design/methodology/approach The survey involved a large sample of 4,667 Italian bank sector’s employees. Findings The results show the invariance of the three dimensional structure of organizational stereotypes towards older workers scale: productivity, reliability and adaptability. Furthermore, the moderation is confirmed: the relationship between organizational age stereotypes and occupational self-efficacy is significant only for older respondents. Research limitations/implications Future studies should aim to replicate the findings with longitudinal designs. Practical implications The study suggests the importance to emphasize the positive characteristics of older workers and to reduce the presence of negative age stereotypes in the workplace, especially in order to foster the occupational self-efficacy of older workers. Originality/value The findings are especially relevant in view of the lack of evidence about the relationship between age stereotypes and occupational self-efficacy.

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

scholarly journals Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

1999 ◽  
Vol 181 (15) ◽  
pp. 4611-4616 ◽  
Author(s):  
Helen D. Simpson ◽  
Frederic Barras
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Cellulose Binding

ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

scholarly journals Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase

1997 ◽  
Vol 321 (3) ◽  
pp. 615-621 ◽  
Author(s):  
Luc BERTRAND ◽  
Didier VERTOMMEN ◽  
Eric DEPIEREUX ◽  
Louis HUE ◽  
Mark H. RIDER ◽  
...  
Keyword(s):  
Adenylate Kinase ◽  
Structural Model ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Multiple Alignment ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Conserved Residues ◽  
Nucleotide Binding Proteins ◽  
Sequence Similarities

Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642Ő9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted.

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