The enzymatic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of ...glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymatic catalysis that extend beyond the bounds of carbohydrate chemistry, but the enzymes themselves play myriad essential roles in diverse biological processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiology and disease, has engendered a large scientific constituency with an interest in glycosidase chemistry. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational analysis of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme–ligand species adjacent to the transition state (or states) and of enzyme–inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mechanical basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative positive charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallographic observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the “electrophilic migration” of the anomeric center along the reaction coordinate. The blossoming of computational approaches, such as ab initio metadynamics, has underscored the quantum mechanical basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymatic synthesis of glycosides, wherein chemists mimic strategies found in nature.
Infant formula milk companies try to develop fortified formula milk that mimics human milk as closely as possible, since it is well-known that breast milk has considerable implications in the ...development of the infant in the first years of life. Human milk is unique in terms of complex oligosaccharides content, known as human milk oligosaccharides (HMOs). Their role in the development of intestinal flora blocking the attachment of pathogens and modulating the immune system of the infant are currently recognized. Due to these biological effects, there is a great interest to introduce the main HMOs in the infant formula milk. Therefore, efficient synthetic strategies for HMOs production are required. Here we present a complete review of HMO production using either (chemo)enzymatic syntheses or cell factory approaches, focusing on the strategies that produce HMOs at least at the milligram scale. 42 HMO structures have already been produced as free sugars. Whereas short HMOs are well obtained by cell factory approaches, complex and branched HMOs are better produced by chemoenzymatic strategies. Inspite of the current advances, production strategies of some biologically relevant HMOs are still missing.
The construction of useful and attainable indicators of fitness assessment deserves special attention in clinical practice. We aimed to construct an indicator of the functional fitness age (FFA) of ...women aged 50 and older by an equation using fitness outcomes and its correlation with chronological age (CA) and to analyze the external validity of our results by comparing our sample to others.
Participants (n = 459, age: 70.3 ± 7.9 years, mean ± SD) were evaluated using the Senior Fitness Test battery. We applied a multiple regression and a subsequent Holt's exponential smoothing to analyze the outcomes.
We obtained a statistically significant expression of F(6, 452) = 328.384; p < 0.0005 in which the coefficients of the equation explain 81% of variability (R2corrected = 0.813). The equation correlates fitness assessment in women aged 50 and over with regards to CA: FFA = 40.146 + 0.350 × CS (stand) − 0.714 × AC (rep) − 0.110 × ST (step) − 0.177 × CSR (cm) − 0.101 × BS (cm) + 8.835 × FUG (s) where CS means chair stand test, AC means arm curl test, ST means 2-min step test, CSR means chair sit-and-reach test, BS means back scratch test, FUG means 8-foot up-and-go test. We compared this index with percentiles distribution from our sample and from other studies.
We suggest the use of FFA as a valid indicator of fitness in adult and senior women as well as a useful motivational tool to undertake exercise programs.
El balonmano es un deporte que requiere la repetición de movimientos y acciones de alta intensidad, como aterrizajes con una pierna y acciones de uno contra uno, que favorecen el mecanismo lesivo del ...ligamento cruzado anterior. El entrenamiento preventivo puede identificar los factores de riesgo neuromusculares asociados al riesgo de sufrir esta lesión en deportistas femeninas. Determinar sus características (duración, frecuencia, tipo de ejercicio…) y componentes (fuerza, pliometría, equilibrio…) es fundamental a la hora de diseñar un entrenamiento que sea específico e individualizado para la jugadora. Los objetivos de este trabajo fueron identificar y categorizar los componentes comunes de los programas de entrenamiento preventivo de la lesión del ligamento cruzado anterior en jugadoras de balonmano y describir y clasificar los ejercicios que conforman cada categoría. Se realizó una revisión sistemática siguiendo las directrices de la declaración PRISMA en las bases de datos Web of Science, Sport Discus, PubMed, Scopus, Cochrane y ScienceDirect. Los criterios de inclusión fueron: (a) las participantes eran jugadoras de balonmano de cualquier edad y sexo femenino, (b) había una intervención con un entrenamiento preventivo, y (c) se informaba de la incidencia lesiva con el número de lesiones de LCA. Se incluyeron seis estudios y se evaluó su calidad metodológica mediante la herramienta ROB 2.0. Los resultados indican que la mayoría de intervenciones incluían más de un componente de entrenamiento con una duración media de 15 minutos y que los ejercicios que más variaban entre los diferentes programas fueron los de pliometría.
The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central ...hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis.
Chitooligosaccharides (COS), the depolymerization products of chitin, have many potential applications in agriculture and medicine since they induce immunostimulating effects and disease protective ...responses. Most of their biological activities require degrees of polymerization (DP) larger than the tetrasaccharide, but structurally well-defined COS with DP larger than six are difficult to produce due to their high insolubility and complex isolation from chitin hydrolysates. Enzymatic synthesis by exploiting the transglycosylation activity of chitinases offers a potential strategy for the assembly of oligomers in the range of bioactive DPs. We here explore the glycosynthase-like activity of six GH18 chitinases from bacterial and archaeal origin by mutating the catalytic assisting residue in the substrate-assisted mechanism of this enzyme family. The alanine mutants at the assisting residue have a significant, but not essential, effect on the hydrolase activity. We studied the ability of the alanine mutants at the assisting residue to catalyze the polymerization of an oxazoline derivative as donor substrate, selecting the oxazoline of pentaacetylchitopentaose (DP5ox) with the aim of obtaining larger oligomers/polymers that, being insoluble, might be resistant to further reactions by the hydrolytically compromised mutant enzymes. For all the enzymes, insoluble polymeric material was obtained, with DP10 as major component, but other COS with different DPs were also obtained, limiting the practical application to produce oligomers/polymers with a defined DP. The balance between the residual hydrolase activity of the mutant enzymes and the solubility/precipitation kinetics still lead to hydrolysis and/or transglycosylation reactions on the newly formed products. From the selected enzymes, the Thermococcus kodakaraensis ChiA D1022A mutant gave the best results, with the formation of insoluble polymers in 45% yield (w/w) and containing about 55% of the target DP10 product.
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•GH18 chitinases operate by substrate-assisted catalysis.•Mutation of the assisting residue has a significant effect on the hydrolase activity.•Mutation of the assisting residue confers glycosynthase-like activity.•An oxazoline derivative donor polymerizes to longer chitin oligomers.•But the residual hydrolase activity leads to a mixture of oligomers.
The glycolipid synthase MG517 from Mycoplasma genitalium catalyzes the glucosyl transfer from UDPGlc to diacylglycerol producing glycoglycerolipids (GGL) (Andrés et al., 2011). The enzyme was ...functional in E. coli accumulating GGL in the plasma membrane. A metabolic engineering strategy for GGL production was evaluated using this microorganism. To increase the levels of GGL precursors, UDPGlc and diacylglycerol, GalU and PlsC enzymes involved in their biosynthesis were overexpressed. Seven engineered strains were obtained containing different combinations of the mg517 with galU and plsC genes. Diacylglycerol synthesis showed to be limiting and the strain overexpressing MG517 and PlsC achieved the highest GGL yield. The new lipids were mono, di- and triglucosyldiacylglycerol with different acyl combinations in each compound. It indicates that the successive glucosyl transferase activities of MG517 have different acyl chain specificity for the acceptor substrate. GGL represented up to 6mg per g of dry weight.
► A metabolic engineering strategy in E. coli for glycolipids production is proposed. ► The MG517 glycosyltransferase from Mycoplasma genitalium is functional in E. coli. ► Strains combining mg517, plsC and galU genes are evaluated as glycolipid producers. ► Overproduction of diacylglycerol precursor, but not UDP-glucose, increases glycoglycerolipids productivity. ► The recombinant strains present a lower proportion of phosphatidylethanolamine in the lipid membrane.
Gait speed is related to physical function in older adults. This cross-sectional study examined the best predictors of maximal gait speed (MGS) among physical abilities, and general factors in ...healthy, rural community-dwelling older adults.
MGS, muscle strength, and postural sway were measured in 55 community-dwelling participants (age, 72.1 ± 6.8, range 61-87 years; 72.7% women). Two stepwise regressions were used to find MGS predictors in two models: physical abilities and global.
Strength of knee extensors with 60° of knee flexion (KStrength60°) and maximal distance in the anterior-posterior direction with eyes closed explained 50.2% of MGS variance (p < .05) in the physical abilities model. KStrength60°, age, and level of physical activity explained 63.9% of MGS variance (p < .05) in the global model.
Regardless of the model, KStrength60° was the best predictor of MGS in rural female older adults. Future research should examine the generalization of these findings to rural male older adults.
Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including ...Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses.
An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from
...Bacillus licheniformis
. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.
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