SNi from SN2: a front-face mechanism ‘synthase’ engineered from a retaining hydrolase

SNi or SNi-like mechanisms, in which leaving group departure and nucleophile approach occur on the same ‘front’ face, have been observed previously experimentally and computationally in both the chemical and enzymatic (glycosyltransferase) substitution reactions of α-glycosyl electrophiles. Given the availability of often energetically comparable competing pathways for substitution (SNi vs SN1 vs SN2) the precise modulation of this archetypal reaction type should be feasible. Here, we show that the drastic engineering of a protein that catalyzes substitution, a retaining β-glycosidase (from Sulfolobus solfataricus SSβG), apparently changes the mode of reaction from “SN2” to “SNi”. Destruction of the nucleophilic Glu387 of SSβG-WT through Glu387Tyr mutation (E387Y) created a catalyst (SSβG-E387Y) with lowered but clear transglycosylation substitution activity with activated substrates, altered substrate and reaction preferences and hence useful synthetic (‘synthase’) utility by virtue of its low hydrolytic activity with unactivated substrates. Strikingly, the catalyst still displayed retaining β stereoselectivity, despite lacking a suitable nucleophile; pH-activity profile, mechanism-based inactivators and mutational analyses suggest that SSβG-E387Y operates without either the use of nucleophile or general acid/base residues, consistent with a SNi or SNi-like mechanism. An x-ray structure of SSβG-E387Y and subsequent metadynamics simulation suggest recruitment of substrates aided by a π-sugar interaction with the introduced Tyr387 and reveal a QM/MM free energy landscape for the substitution reaction catalyzed by this unnatural enzyme similar to those of known natural, SNi-like glycosyltransferase (GT) enzymes. Proton flight from the putative hydroxyl nucleophile to the developing p-nitrophenoxide leaving group of the substituted molecule in the reactant complex creates a hydrogen bond that appears to crucially facilitate the mechanism, mimicking the natural mechanism of SNi-GTs. An oxocarbenium ion-pair minimum along the reaction pathway suggests a step-wise SNi-like DN*ANss rather than a concerted SNi DNAN mechanism. This first observation of a front face mechanism in a β-retaining glycosyl transfer enzyme highlights, not only that unusual SNi reaction pathways may be accessed through direct engineering of catalysts with suitable environments, but also suggests that ‘β-SNi’ reactions are also feasible for glycosyl transfer enzymes and the more widespread existence of SNi or SNi-like mechanism in nature

Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors

Methodological strategies to understand smartphone practices for social connectedness in later life

Digital practices in later life are not yet well understood. Therefore, this paper discusses the framework for a research design project that aims at tracing differences and similarities in how older adults use their smartphones in circumstances in and outside their homes in Spain, the Netherlands, Sweden, and Canada. The research questions of this international research project focus on the extent to which digital mobile practices relate to perceived social connectedness among older adults aged 55–79 years old. While studies have shown that the subjective experience of ‘being connected’ supports continued wellbeing in later life, there remains an insufficient understanding of the processes through which digital mediated social interaction is effective for social connectedness. The analytical framework of the project prioritizes the co-constituency of (digital) technology and ageing, and takes digital practices in everyday life as its entry point. The main data collection tool will be the tracking of smartphone activity of 600 older adults (150 per country) during four weeks. An online survey and qualitative interviews will gather data about the meanings of the quantified digital practices, and how they shape (if they do) the participants’ connection to the world. This approach will allow us not only to get insight into what older adults say how they used their smartphone but also to gain insight into their real-life daily use. The assessment of the challenges, strengths, and weaknesses of the methods contributes towards an accurate and appropriate interpretation of empirical results and their implications

Older audiences in the digital media environment: A cross-national longitudinal study:Wave 1 Report 1.0

Involving teams from seven countries (Austria, Canada, Denmark, Israel, Netherlands, Romania, and Spain) this Ageing + Communication + Technologies (ACT) project offers a unique opportunity to explore possible processes of displacement of traditional dominant media by innovative communication practices within the older audience of new media. Replicating Nimrod’s (2017) study of older audiences, data will be collected on a biannual basis over a five-year period (overall three waves). The first wave was based on surveys with Internet users aged 60 and up, to whom we will return in the following waves. Data was collected by local commercial firms. With the exception of Romania, where the survey was conducted via telephone due to a low rate of Internet users among the older population, all firms applied an online survey. Most data was collected between November and December 2016, with the exception of Canada, where the data were collected between June and July 2017. With varying expected dropout rates, the samples were planned to have a final panel that will comprise about 500 participants per country. For this reason, sample sizes in the first wave were not equal and ranged between 715 (Denmark) and 3,538 (Canada). The overall sample size consisted of 10,527 Internet users aged 60 and over. To reach this sample size, the firms contacted a total of 33,035 individuals. Response rates ranged between 8.9% and 64.6% according to percentage of older people who use the Internet in the country and data collection method. The response rate was at its lowest in Romania, where there was a need to screen out older adults who do not use the Internet, and at its highest in the Netherlands

New radiometric dates for the Prehistory of Northwestern Iberia: Valdavara Cave (Becerreá, Lugo)

The goal of this paper is to make known the first results of the excavations in Valdavara Cave (Becerreá, Lugo), paying special attention to the radiometric dating that places this cave among the few Galician sites with Upper Palaeolithic dates. During the excavation carried out in 2007, two archaeological locations were documented in the same karstic system (Valdavara 1 and Valdavara 2). The test pit initiated in Valdavara 1 allowed us to identify so far two stratigraphic units: an upper unit corresponding to Recent Prehistory and a lower unit (level 4) that yielded the Upper Palaeolithic dates. An assemblage of human remains from the Bronze Age was found in Valdavara 2.<br><br>El objetivo de este artículo es dar a conocer los primeros resultados de las excavaciones en la cueva de Valdavara (Becerreá, Lugo), prestando especial atención a las dataciones radiométricas que la sitúan entre los pocos yacimientos gallegos que han arrojado fechas correspondientes al Paleolítico Superior. La excavación realizada en el año 2007 permitió documentar dos localizaciones arqueológicas en el marco del mismo sistema cárstico (Valdavara 1 y Valdavara 2). El sondeo iniciado en Valdavara 1 ha permitido identificar hasta el momento dos conjuntos estratigráficos: un conjunto superior correspondiente a la Prehistoria reciente y un conjunto inferior (nivel 4) en el que se han obtenido las fechas del Paleolítico Superior final. En Valdavara 2 se encontró un conjunto de restos humanos de la Edad del Bronce