Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed β-3-prism scaffold.

Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed β-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 β-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains.

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source.

Glutaminase Affects the Transcriptional Activity of Peroxisome Proliferator-Activated Receptor γ (PPARγ) via Direct Interaction.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle.

N-terminal phosphorylation of glutaminase C decreases its enzymatic activity and cancer cell migration.

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function.

The dihydroorotate dehydrogenases: Past and present.

The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs.

The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis.

A thermostable variant of the mesophilic xylanase A from Bacillus subtilis (BsXynA-G3_4x) contains the four mutations Gln7His, Gly13Arg, Ser22Pro, and Ser179Cys. The crystal structure of the BsXynA-G3_4x has been solved, and the local environments around each of these positions investigated by molecular dynamics (MD) simulations at 328K and 348K. The structural and MD simulation results were correlated with thermodynamic data of the wild-type enzyme, the 4 single mutants and the BsXynA-G3_4x.

Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom.

BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry.

Target sites for the design of anti-trypanosomatid drugs based on the structure of dihydroorotate dehydrogenase.

Trypanosomatids consist of a large group of flagellated parasitic protozoa, including parasites from the genera Leishmania and Trypanosoma, responsible for causing infections in millions of humans worldwide and for which currently no appropriate therapy is available. The significance of pyrimidines in cellular metabolism makes their de novo and salvage pathways ideal druggable targets for pharmacological intervention and open an opportunity for pharmaceutical innovation. In the current review, we discuss the merits in targeting the enzyme dihydroorotate dehydrogenase (DHODH), a flavin-dependent enzyme that catalyzes the fourth and only redox step in pyrimidine de novo biosynthesis, as a strategy for the development of efficient therapeutic strategies for trypanosomatid-related diseases.

Crystal structure of dihydroorotate dehydrogenase from Leishmania major.

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs.

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida.

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops.

Novel insights for dihydroorotate dehydrogenase class 1A inhibitors discovery.

The enzyme dihydroorotate dehydrogenase (DHODH) has been suggested as a promising target for the design of trypanocidal agents. We report here the discovery of novel inhibitors of Trypanosoma cruzi DHODH identified by a combination of virtual screening and ITC methods. Monitoring of the enzymatic reaction in the presence of selected ligands together with structural information obtained from X-ray crystallography analysis have allowed the identification and validation of a novel site of interaction (S2 site).

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the N-terminal carbohydrate-recognition domain of human galectin-4.

Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on.

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain.

Trypanosoma cruzi is the etiological agent of Chagas' disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T. cruzi strain Y solved at 2.