Concept: Enzyme substrate
The specificity for the α-1,4- and α-1,6-glucosidic linkages varies among glycoside hydrolase family 31 α-glucosidases. This difference in substrate specificity has been considered to be due to the difference in an aromatic residue on β→α loop 1 in the catalytic domain with a (β/α)8 barrel fold; i.e., the enzymes having Tyr and Trp on β→α loop 1 were respectively described as α-1,4-specific and α-1,6-specific α-glucosidases. Schwanniomyces occidentalis α-glucosidase, however, prefers the α-1,4-glucosidic linkage, although the enzyme possesses Trp324 at the corresponding position. The mutation of Trp324 to Tyr decreased the ability for hydrolysis of the α-1,6-glucosidic linkage and formation of the α-1,6-glucosidic linkage in transglycosylation, indicating Trp324 to be closely associated with α-1,6 specificity, even if the enzyme preferred the α-1,4-glucosidic linkage. The mutant enzyme was found to catalyze the production of the branched oligosaccharide, 2,4-di-O-(α-D-glucopyranosyl)-D-glucopyranose, more efficiently than the wild-type enzyme.
The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system’s peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.
Underwater locomotion in a terrestrial beetle: combination of surface de-wetting and capillary forces.
- Proceedings. Biological sciences / The Royal Society
- Published over 7 years ago
For the first time, we report the remarkable ability of the terrestrial leaf beetle Gastrophysa viridula to walk on solid substrates under water. These beetles have adhesive setae on their feet that produce a secretory fluid having a crucial role in adhesion on land. In air, adhesion is produced by capillary forces between the fluid-covered setae and the substrate. In general, capillary forces do not contribute to adhesion under water. However, our observations showed that these beetles may use air bubbles trapped between their adhesive setae to walk on flooded, inclined substrata or even under water. Beetle adhesion to hydrophilic surfaces under water was lower than that in air, whereas adhesion to hydrophobic surfaces under water was comparable to that in air. Oil-covered hairy pads had a pinning effect, retaining the air bubbles on their feet. Bubbles in contact with the hydrophobic substrate de-wetted the substrate and produced capillary adhesion. Additional capillary forces are generated by the pad’s liquid bridges between the foot and the substrate. Inspired by this idea, we designed an artificial silicone polymer structure with underwater adhesive properties.
Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a ‘hotspot’ on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon ‘neosubstrate’ selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.
L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.
OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.
LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze polyketide chain initiation reaction using isobutyryl-CoA as a substrate, and elongate once with methylmalonyl-CoA to start the biosynthesis of antibiotic α-lipomycin in Streptomyces aureofaciens Tü117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs including isobutyryl-CoA were accepted as the starter substrates while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.
Lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) (bsLDH) has a crucial role in producing chirally pure hydroxyl compounds. α-Hydroxy acids are used in many industrial situations, ranging from pharmaceutical to cosmetic dermatology products. One drawback of this enzyme is its limited substrate specificity. For instance, l-lactate dehydrogenase exhibits no detectable activity towards the large side chain of 2-hydroxy acid l-mandelic acid, an α-hydroxy acid with anti-bacterial activity. Despite many attempts to engineer bsLDH to accept α-hydroxy acid substrates, there have been no attempts to introduce the industrially important l-mandelic acid to bsLDH. Herein, we describe attempts to change the reactivity of bsLDH towards l-mandelic acid. Using the Insight II molecular modelling programme (except ‘program’ in computers) and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, T246G and I240A, would allow the enzyme to utilise l-mandelic acid as a substrate. Genes encoding for the wild-type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in Escherichia coli and purified using the TAGZyme system. Enzyme assays showed that insertion of this double mutation into highly purified bsLDH switched the substrate specificity from lactate to l-mandelic acid.
Commercial inulinase from Aspergillus niger was immobilized in montmorillonite and then treated in pressurized propane and liquefied petroleum gas (LPG). Firstly, the effects of system pressure, exposure time, and depressurization rate, using propane and LPG, on enzymatic activity were evaluated through central composite design 2(3). Residual activities of 145.1 and 148.5 % were observed for LPG (30 bar, 6 h, and depressurization rate of 20 bar min(-1)) and propane (270 bar, 1 h, and depressurization rate of 100 bar min(-1)), respectively. The catalysts treated at these conditions in both fluids were then used for the production of fructooligosaccharides (FOS) using sucrose and inulin as substrates in aqueous and organic systems. The main objective of this step was to evaluate the yield and productivity in FOS, using alternatives for enhancing enzyme activity by means of pressurized fluids and also using low-cost supports for enzyme immobilization, aiming at obtaining a stable biocatalyst to be used for synthesis reactions. Yields of 18 % were achieved using sucrose as substrate in aqueous medium, showing the potential of this procedure, hence suggesting a further optimization step to increase the process yield.
Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.