130,566 research outputs found
Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase
No full textLimited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase
The PHBH fold: not only flavoenzymes
No full textp-Hydroxybenzoate hydroxylase, D-amino acid oxidase, cholesterol oxidase and glucose oxidase form a family of structurally related flavoenzymes. Comparison of their three-dimensional structures reveal how the same FAD-binding scaffold has been employed to implement diverse active-site architectures, suited for different types of catalytic reactions. The substrate binding mode differs in each of these enzymes, with the catalytically relevant residues not located on homologous positions. A common feature is provided by the ability of these enzyme to bury their substrates beneath the protein surface. In D-amino acid oxidase and cholesterol oxidase, a loop forms a 'lid' controlling the active site accessibility, whereas in p-hydroxybenzoate hydroxylase is the flavin itself, which swings out to allow substrate binding. The crystallographic analysis has revealed that the GTP-dissociation inhibitor of RAB GTPases has a folding topology remarkably similar to p-hydroxybenzoate hydroxylase. This finding highlights the versatile nature of this folding topology, which in addition to flavin-dependent catalysis, is suited for diverse functions, such as the regulation of GTPases
La misura del dubbio: giustizia riparativa ex d. lgs. n. 150/2022 e d.lgs. n. 231/2001
No full textIl contributo analizza l'impatto del sistema della disciplina organica della giustizia riparativa sulla normativa relativa alla responsabilità d'impresa ex d.lgs. 231/200
Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis
No full textL-Aspartate oxidase (Laspo) catalyzes the conversion of L-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD +. This bacterial pathway represents a potential drug target since it is absent in mammals. The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 Å resolution in both the native state and in complex with succinate. Comparison of the R386L holoprotein with the wild-type apoenzyme [Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S. (1999) Structure 7, 745-756] reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44-53 and 104-141) and to a 27° rotation of the capping domain. This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain. The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates. Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the L-Asp α-amino group. The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family. This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical D-amino acid oxidase
Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis
No full textL-Aspartate oxidase (Laspo) catalyzes the conversion of L-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD(+). This bacterial pathway represents a potential drug target since it is absent in mammals. The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 A resolution in both the native state and in complex with succinate. Comparison of the R386L holoprotein with the wild-type apoenzyme [Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S. (1999) Structure 7, 745-756] reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44-53 and 104-141) and to a 27 degree rotation of the capping domain. This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain. The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates. Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the L-Asp alpha-amino group. The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family. This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical D-amino acid oxidase
Structure of D-amino acid oxidase: new insights from an old enzyme
No full textD-amino acid oxidase is the prototype of flavin-dependent oxidases. The recent resolution of its 3D structure has provided an explanation for several of its properties and has led to a substantial revision of the mechanism of D-amino acid dehydrogenation, with significant implications for the general understanding of flavin-dependent catalysis
Mechanistic Aspects Regarding the Elimination of H2O2 from C(4a)-Hydroperoxyflavin. The Role of a Proton Shuttle Required for H2O2 Elimination.
No full textDFT calculations presented for C(4a)-hydroperoxyflavin (C(4a)-FLHOOH) at the B3LYP/6-311+G(d,p) level suggest a new mechanism for the elimination of H2O2. The calculated activation barrier for a concerted four-centered elimination (DeltaE() = 32.86 kcal/mol) strongly suggests that in the absence of interactions with the local environment a spontaneous elimination is not feasible. A proton shuttle from the N5 hydrogen to the proximal oxygen of the OOH moiety involving three water molecules has an activation barrier that is reduced to 17.11 kcal/mol. Calculations that utilize CH3OH to model the role of a local Thr or Ser residue shows that an alcohol functionality hydrogen bonded to the N5 H-atom can catalyze the elimination of H2O2 with a free energy of activation of 21.5 kcal/mol. Interaction of amines and amide residues (CH3NH2 and CH3(C═O)NH2) with the N5 locus of C(4a)-hydroperoxyflavin markedly reduce the activation barrier for H2O2 elimination relative to the concerted pathway. Proton transfer from a COOH group (DeltaG() = 8.36 kcal/mol) or the NH2 group of a positively charged Arg model (DeltaG() = 9.99 kcal/mol) to the proximal oxygen of the OOH moiety of C(4a)-FLHOOH in the TS for H2O2 elimination strongly enhances elimination of H2O
Active site Plasticity in D-aminoacid Oxidase; a crystallographic analysis
No full textD-Amino acid oxidase (DAAO) is the prototype of the flavin-containing oxidases. It catalyzes the oxidative deamination of various D-amino acids, ranging from D-Ala to D-Trp. We have carried out the X-ray analysis of reduced DAAO in complex with the reaction product imino tryptophan (iTrp) and of the covalent adduct generated by the photoinduced reaction of the flavin with 3-methyl-2-oxobutyric acid (kVal). These structures were solved by combination of 8-fold density averaging and least-squares refinement techniques. The FAD redox state of DAAO crystals was assessed by single-crystal polarized absorption microspectrophotometry. iTrp binds to the reduced enzyme with the N, C alpha, C, and C beta atoms positioned 3.8 A from the re side of the flavin. The indole side chain points away from the cofactor and is bound in the active site through a rotation of Tyr224. This residue plays a crucial role in that it adapts its conformation to the size of the active site ligand, providing the enzyme with the plasticity required for binding a broad range of substrates. The iTrp binding mode is fully consistent with the proposal, inferred from the analysis of the native DAAO structure, that substrate oxidation occurs via direct hydride transfer from the C alpha to the flavin N5 atom. In this regard, it is remarkable that, even in the presence of the bulky iTrp ligand, the active center is made solvent inaccessible by loop 216-228. This loop is thought to switch between the "closed" conformation observed in the crystal structures and an "open" state required for substrate binding and product release. Loop closure is likely to have a role in catalysis by increasing the hydrophobicity of the active site, thus making the hydride transfer reaction more effective. Binding of kVal leads to keto acid decarboxylation and formation of a covalent bond between the keto acid C alpha and the flavin N5 atoms. Formation of this acyl adduct results in a nonplanar flavin, characterized by a 22 degrees angle between the pyrimidine and benzene rings. Thus, in addition to an adaptable substrate binding site, DAAO has the ability to bind a highly distorted cofactor. This ability is relevant for the enzyme's function as a highly efficient oxidase
New structures of allosteric proteins revealing remarkable conformational changes
No full textNew three-dimensional structures of allosteric proteins reveal they have a flexible architecture that is instrumental to the regulation of protein function. Highlights are the structures of GroEL, pyruvate kinase, D-3-phosphoglycerate dehydrogenase and the acetylcholine receptor. Furthermore, significant progress in understanding the nature of the intermediates involved in an allosteric reaction has been achieved through recent spectroscopic and crystallographic studies on haemoglobin
MeSH term explosion and author rank improve expert recommendations
No full textInformation overload is an often-cited phenomenon that reduces the productivity, efficiency and efficacy of scientists. One challenge for scientists is to find appropriate collaborators in their research. The literature describes various solutions to the problem of expertise location, but most current approaches do not appear to be very suitable for expert recommendations in biomedical research. In this study, we present the development and initial evaluation of a vector space model-based algorithm to calculate researcher similarity using four inputs: 1) MeSH terms of publications; 2) MeSH terms and author rank; 3) exploded MeSH terms; and 4) exploded MeSH terms and author rank. We developed and evaluated the algorithm using a data set of 17,525 authors and their 22,542 papers. On average, our algorithms correctly predicted 2.5 of the top 5/10 coauthors of individual scientists. Exploded MeSH and author rank outperformed all other algorithms in accuracy, followed closely by MeSH and author rank. Our results show that the accuracy of MeSH term-based matching can be enhanced with other metadata such as author rank
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