Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or β-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a β-propeller catalytic domain, and a β-sandwich domain with unknown function.

Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification.

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions.

Insights into the Structure of the Highly Glycosylated Ffase from Enhance Its Biotechnological Potential.

β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast.

Structure-Function Insights into the Fungal -Chitinase Chit33 Depict its Mechanism on Chitinous Material.

Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type -chitinase Chit33 from and its D165A/E167A-Chit33-(NAG) complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions.

Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism.

Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from , which represent different states along the enzymatic mechanism.

Phylogenetic, functional and structural characterization of a GH10 xylanase active at extreme conditions of temperature and alkalinity.

Endoxylanases active under extreme conditions of temperature and alkalinity can replace the use of highly pollutant chemicals in the pulp and paper industry. Searching for enzymes with these properties, we carried out a comprehensive bioinformatics study of the GH10 family. The phylogenetic analysis allowed the construction of a radial cladogram in which protein sequences putatively ascribed as thermophilic and alkaliphilic appeared grouped in a well-defined region of the cladogram, designated TAK Cluster.

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis.

The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 studied to explore their role in modulating this biocatalytic process.

Crocetin Isolated from the Natural Food Colorant Saffron Reduces Intracellular Fat in 3T3-L1 Adipocytes.

Saffron, as a food colorant, has been displaced by low-cost synthetic dyes. These have unhealthy properties; thus, their replacement with natural food colorants is an emerging trend. Obesity is a worldwide health problem due to its associated comorbidities.

Structural analysis of the reducing-end xylose-releasing exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 deciphers its molecular specificity.

Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillus barcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides.

The cryo-EM Structure of β-Galactosidase: Quaternary Structure Guides Protein Engineering.

Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population. The severity of the symptoms is highly variable, depending on the susceptibility to the sugar and the amount digested. For that reason, enzymes that can be used for the production of lactose-free milk and milk derivatives have acquired singular biotechnological importance.

Structural basis of the inhibition of GH1 β-glucosidases by multivalent pyrrolidine iminosugars.

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 β-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes.

Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity.

Background: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units.