Rational engineering of a hyperstable glycosyltransferase for blue denim dyeing

Indigo is one of the most used dyes to produce the textile blue denim worldwide1. Its synthesis and the dyeing process require chemical steps that are environmentally damaging, including the use of reducing agents for indigo solubilization. The glycosyltransferase PtUGT1 is able to add a glucose moiety to the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation and keeping it soluble2. This strategy could be used in a chemoenzymatic approach to replace the use of reducing agents, but in order to do this it’s necessary that PtUGT1 resist, and to be active under the harsh conditions used in the industrial process, including high pH and high temperature. We have characterized the activity profile of PtUGT1 at different pH values and temperatures and have determined the enzyme stability by differential scanning fluorometry (DSF) and residual activity. Leveraging the structure information of PtUGT1 obtained by X-ray crystallography (PDB ID: 5nlm)2, we have rationally designed different mutants to develop a variant adapted to higher temperatures and pH values, including hypothetical residue pair mutants that could lead to the formation of intramolecular disulfide bridges, and mutants that could either improve the hydrophobic packing, lead to formation of polar interactions or improve Pro/Gly ratio, consequently increasing the rigidity/stability of PtUGT1. As a result we have developed several active PtUGT1 variants with up to 15°C increase in their melting temperature (TmB) (Fig. 1), the highest ever reported for an UDP-dependent glycosyltransferase. Please click Additional Files below to see the full abstract

Lytic polysaccharide monooxygenases:a crystallographer's view on a new class of biomass-degrading enzymes

Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future

Characterization of different crystal forms of the α-glucosidase MalA from \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

MalA is an _-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting α-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P21, from which data sets extending to 2.5 Å resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P212121) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry

Characterization of different crystal forms of the α-glucosidase MalA from \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

MalA is an _-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting α-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P21, from which data sets extending to 2.5 Å resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P212121) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry

Expression, refolding and spectroscopic characterization of fibronectin type III (FnIII)-homology domains derived from human fibronectin leucine rich transmembrane protein (FLRT)-1,-2, and-3

The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both cis- and trans-interactions, whereas the membrane proximal part is a fibronectin type III (FnIII) domain responsible for a cis-interaction with members of the fibroblast growth factor receptor 1 (FGFR1) family, which results in FGFR tyrosine kinase activation. Whereas the structures of FLRT LRR domains from various species have been determined, the expression and purification of recombinant FLRT FnIII domains, important steps for further structural and functional characterizations of the proteins, have not yet been described. Here we present a protocol for expressing recombinant FLRT-FnIII domains in inclusion bodies in Escherichia coli. His-tags permitted affinity purification of the domains, which subsequently were refolded on a Ni-NTA agarose column by reducing the concentration of urea. The refolding was confirmed by circular dichroism (CD) and 1H-NMR. By thermal unfolding experiments we show that a strand-strand cystine bridge has significant effect on the stability of the FLRT FnIII fold. We further show by Surface Plasmon Resonance that all three FnIII domains bind to FGFR1, and roughly estimate a Kd for each domain, all Kds being in the µM range

A hyperstable glycosyltransferase for blue denim dyeing

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Structural characterization of the thermostable <i>Bradyrhizobium japonicum</i> D-sorbitol dehydrogenase

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is d-glucitol (a synonym for d-sorbitol), although l-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with d-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T (m) for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio