GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

Structural insights into sialic acid enzymology

Sialic acids are a diverse family of negatively charged sugars that play essential biological roles. Their presence and relative abundance in different cells is ultimately regulated by the concerted action of a large set of enzymes. In this review, we focus on the most recent advances on the enzymes that govern sialic acid metabolism, with emphasis on structural work. Major progress has been made in dissecting the catalytic mechanism of sialidases, revealing a modified scenario of the typical glycosidase ping-pong mechanism. Similarly, X-ray structures of sialyltransferases uncover significant variations of formerly known glycosyltransferase foldings. Both sialidases and sialyltransferases seem to tell us that sialic acid-handling enzymes have evolved important modifications related to the distinctive features of sialic acid itself.     

Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC₅₀ values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.     

Optimization of the dilute maleic acid pretreatment of wheat straw

Background  

In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.     

Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Sialidases are a superfamily of sialic-acid-releasing enzymes that are of significant interest due to their implication as virulence factors in the pathogenesis of a number of diseases. However, extensive studies of viral and microbial sialidases have failed to provide a comprehensive picture of their mechanistic properties, in part because the structures of competent enzyme-substrate complexes and reaction intermediates have never been described. Here we report these structures for the Trypanosoma cruzi trans-sialidase (TcTS), showing that catalysis by sialidases occurs via a similar mechanism to that of other retaining glycosidases, but with some intriguing differences that may have evolved in response to the substrate structure.     

Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects.

The internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, Ile, Tyr) by site-directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA- background. The substitution F25A severely impaired the functional properties of the enzyme. Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45-60% that of the wild type at 37 degrees, and essentially identical at 42 degrees. At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies. In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37 degrees, demonstrating that subtle modifications of the protein interior at position 25 Ile/Leu or Phe/Tyr) can produce significant biological effects. All F25 mutants were good substrates for E. coli thioredoxin reductase. Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates. Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability. Thus, thermodynamic stability of R. sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro.     

The carboxy-terminal C2-like domain of the α-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition

The lethal, cytolytic α-toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N-terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C-terminal domain. Calcium ions are essential for the binding of α-toxin to lipid films. Sixteen α-toxin variants with single amino acid substitutions in the C-terminal region were obtained using site-directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were considerably less active than native α-toxin under calcium-limiting conditions. Replacement of Thr-272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface-exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C₂β-barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. The combined findings suggest that the C-terminal region of α-toxin mediates interactions with membrane phospholipids in a calcium-dependent manner. Mutations to this domain may account for the natural lack of toxicity of the α-toxin homologue, phospholipase C of Clostridium bifermentans .     

Studies of structure and specificity of some antigen-antibody complexes

By using X-ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen-antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.     

Three-dimensional structure determination of an anti-2-phenyloxazolone antibody: the role of somatic mutation and heavy/light chain pairing in the maturation of an immune response.

The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family.