1,720,961 research outputs found
Serine hydroxymethyltransferase: A model enzyme for mechanistic, structural, and evolutionary studies.
No full textSerine hydroxymethyltransferase is a ubiquitous representative of the family of fold type I, pyridoxal 5'-phosphate-dependent enzymes. The reaction catalyzed by this enzyme, the reversible transfer of the Cβ of serine to tetrahydropteroylglutamate, represents a link between amino acid and folates metabolism and operates as a major source of one-carbon units for several essential biosynthetic processes. Serine hydroxymethyltransferase has been intensively investigated because of the interest aroused by the complex mechanism of the hydroxymethyltransferase reaction and its broad substrate and reaction specificity. Although the increasing availability of crystallographic data and the characterization of several site-specific mutants helped in understanding previous functional and structural studies, they also represent the starting point of novel investigations. This review will focus on recently highlighted catalytic, structural, and evolutionary aspects of serine hydroxymethyltransferase. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology
Alanine racemase from Tolypocladium inflatum: A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity
No full textCyclosporin A, a cyclic peptide produced by the fungus Tolypocladium inflatum, is a widely employed immunosuppressant drug. Its biosynthesis is strictly dependent on the action of the pyridoxal 5'-phosphate-dependent enzyme alanine racemase, which produces the D-alanine incorporated in the cyclic peptide. This enzyme has a different fold with respect to bacterial alanine racemases. The interest elicited by T. inflatum alanine racemase not only relies on its biotechnological relevance, but also on its evolutionary and structural similarity to the promiscuous enzymes serine hydroxymethyltransferase and threonine aldolase. The three enzymes represent a model of divergent evolution from an ancestral enzyme that was able to catalyse all the reactions of the modern enzymes. A protocol to express and purify with high yield recombinant T. inflatum alanine racemase was developed. The catalytic properties of the enzyme were characterized. Similarly to serine hydroxymethyltransferase and threonine aldolase, T. inflatum alanine racemase was able to catalyse retroaldol cleavage and transamination reactions. This observation corroborates the hypothesis of the common evolutionary origin of these enzymes. A three-dimensional model of T. inflatum alanine racemase was constructed on the basis of threonine aldolase crystal structure. The model helped rationalise the experimental data and explain the catalytic properties of the enzymes. (C) 2012 Elsevier Inc. All rights reserved
On the catalytic mechanism and stereospecificity of Escherichia coli L-threonine aldolase
No full textL-Threonine aldolases (L-TAs) represent a family of homologous pyridoxal 5′-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several l-3-hydroxy-α-amino acids. L-TAs have great biotechnological potential, as they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for the bioorganic synthesis of l-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many L-TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, L-TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low-specificity L-TA from Escherichia coli at 2.2Å resolution, in the unliganded form and cocrystallized with l-serine and l-threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro-aldol cleavage reaction. Interestingly, the very large active site opening of E. Coli L-TA suggests that much larger molecules than L-threonine isomers may be easily accommodated, and L-TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues
Role of a Conserved Active Site Cation pi Interaction in Escherichia coli Serine Hydroxymethyltransferase
No full textSerine hydroxymethyltransferase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of serine and glycine using tetrahydropteroylglutamate as the one-carbon carrier. In all pyridoxal phosphate-dependent enzymes, amino acid substrates are bound and released through a transaldimination process, in which an internal aldimine and an external aldimine are interconverted via gem-diamine intermediates. Bioinformatic analyses of serine hydroxymethyltransferase sequences and structures showed the presence of two highly conserved residues, a tyrosine and an arginine, engaged in a cation-pi interaction. In Escherichia coli serine hydroxymethyltranferase, the hydroxyl group of this conserved tyrosine (Tyr55) is located in a position compatible with a role as hydrogen exchanger in the transaldimination reaction. Because of the location of Tyr55 at the active site, the enhancement of its acidic properties caused by the cation-pi interaction with Arg235, and the hydrogen bonds established by its hydroxyl group, a role of this residue as acid-base catalyst in the transaldimination process was envisaged. The role played by this cation-pi interaction in the E. coli serine hydroxymethyltransferase was investigated by crystallography and site-directed mutagenesis using Y55F and three R235 mutant forms. The crystal structure of the Y55F mutant suggests that the presence of Tyr55 is indispensable for a correct positioning of the cofactor and for the maintenance of the structure of several loops involved in substrate and cofactor binding. The kinetic properties of all mutant enzymes are profoundly altered. Substrate binding and rapid kinetic experiments showed that both Y55 and R235 are required for a correct progress of the transaldimination reactio
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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