Effects of the magnesium and chloride ions and shikimate on the structure of shikimate kinase from Mycobacterium tuberculosis

Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.

Structure, ferroelectric/magnetoelectric properties and leakage current density of (Bi0.85Nd0.15)FeO3 thin films

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mimics Epstein-Barr virus EBNA1 immune evasion through central repeat domain effects on protein processing

Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pyrearinus termitilluminans larval click beetle luciferase: Active site properties, structure and function relationships and comparison with other beetle luciferases

Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, KM values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging. © The Royal Society of Chemistry and Owner Societies 2009.

Structure of a novel class II phospholipase D: Catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7. Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference. © 2011 Elsevier Inc.

Aspergillus niger beta-Glucosidase Has a Cellulase-like Tadpole Molecular Shape INSIGHTS INTO GLYCOSIDE HYDROLASE FAMILY 3 (GH3) beta-GLUCOSIDASE STRUCTURE AND FUNCTION

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Studies of Author Cocitation Analysis: A Bibliometric Approach for Domain Analysis

Author Cocitation Analysis (ACA) can be defined as the analysis of a group of actors, writers or researchers structurally organized in a (social and cognitive) network of a particular scientific community. The greater the number of researchers selected, the greater the amplitude and the domain boundary under consideration; the more restricted the number of researchers chosen as representative and appropriate, the less extensive the domain. From the perspective of the first axis of Tennis (2003), the selection of authors involves setting parameters on the extent of the domain, i.e., its total scope and amplitude. Thus, from the point of view of Tennis’s (2003) approach to Domain Analysis, the selection of authors for Author Cocitation Analysis is associated with the designations and boundaries of the domain, as well as to their goals (Tennis, 2003). Still, the selection of authors through the most cited authors in the literature, reflects the core elements of a domain and constitute the most specific foundation of a domain, aligning to the Degrees of Specialization characterized by Tennis (2003). It is concluded that the Author Cocitation Analysis (ACA) is a relevant procedure to the analysis of the underlying structure of a scientific knowledge domain, which meets the theories and concepts of Domain Analysis researchers, in that it allows characterizing the science, identifying, analyzing and assessing the conditions under which scientific knowledge is constructed and socialized.

RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)