Structural Insights and Reaction Profile of a New Unspecific Peroxygenase from Produced in a Tandem-Yeast Expression System.

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from (UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications.

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.

Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor.

Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.

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.

The Mobility of the Cap Domain Is Essential for the Substrate Promiscuity of a Family IV Esterase from Sorghum Rhizosphere Microbiome.

Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the environmental DNA (eDNA) was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH, retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates.

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity.

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.

Crystal structure of a family VIII β-lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope.

Family VIII esterases present similarities to class C β-lactamases, which show nucleophilic serines located at the S-X-X-K motif instead of the G-X-S-X-G or G-D-S-(L) motif shown by other carboxylesterase families. Here, we report the crystal structure of a novel family VIII (subfamily VIII. I) esterase (EH ; denaturing temperature, 52.

The bone-degrading enzyme machinery: From multi-component understanding to the treatment of residues from the meat industry.

Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm , and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.

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.

Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping.

The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported.

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.

Tuning the Properties of Natural Promiscuous Enzymes by Engineering Their Nano-environment.

Owing to their outstanding catalytic properties, enzymes represent powerful tools for carrying out a wide range of (bio)chemical transformations with high proficiency. In this context, enzymes with high biocatalytic promiscuity are somewhat neglected. Here, we demonstrate that a meticulous modification of a synthetic shell that surrounds an immobilized enzyme possessing broad substrate specificity allows the resulting nanobiocatalyst to be endowed with enantioselective properties while maintaining a high level of substrate promiscuity.

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.

Catalytic Cycle of Glycoside Hydrolase BglX from and Its Implications for Biofilm Formation.

BglX is a heretofore uncharacterized periplasmic glycoside hydrolase (GH) of the human pathogen . X-ray analysis identifies it as a protein homodimer. The two active sites of the homodimer comprise catalytic residues provided by each monomer.

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.

Deciphering the molecular specificity of phenolic compounds as inhibitors or glycosyl acceptors of β-fructofuranosidase from Xanthophyllomyces dendrorhous.

Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV).

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.

Structural Insights into the Substrate Promiscuity of a Laboratory-Evolved Peroxygenase.

Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C-H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å.

Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose.

The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S.