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

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Genomic reconstruction of the SARS-CoV-2 epidemic in England.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021