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Taking PAR apart Proteins can be reversibly modified through the addition of repeating, polymerized ADP-ribose (PAR) subunits catalysed by poly(ADP-ribose) polymerase (PARP). Removal of PAR requires a glycohydrolase (PARG), which cleaves the ribose–ribose bond between subunits. Ivan Ahel and colleagues report that bacteria and fungi have a divergent PARG, which is unrelated to other enzymes that cleave PAR. Its structure, in complex with ADP-ribose and with a PARG inhibitor, and the results of mutational analysis suggest that the mechanism used in mammals and bacteria may be conserved. PARP inhibitors are being developed as pharmaceuticals for diseases including cancer, and this work suggests that small, cell-permeable PARG inhibitors might also be possible drug candidates. Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis 1 . Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases 1 , 2 . PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death 3 , 4 . Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata ), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family 5 , 6 . High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Structural characterization of carboxylic acid reductase in multiple didomain configurations, coupled with mutagenesis, reveals how the enzyme transitions into a catalytically competent orientation and prevents over-reduction of its aldehyde product. Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation–PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP–reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that ( R )-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo -glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo -mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo -glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo -mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios. Poly-ADP-ribosylation is a post-translational modification that is countered by poly(ADP-ribose) glycohydrolases (PARGs). In this study, the authors present the crystal structure of poly(ADP-ribose) glycohydrolase (PARGs) in complex with a poly(ADP-ribose) substrate, and reveal that poly(ADP-ribose) glycohydrolase (PARGs) enzymes act predominantly as exo - rather than as endo -glycohydrolases.

The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic \"nonnatural\" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.

Summary CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid‐based systems for genome editing (gene knockouts/knock‐ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR‐Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR‐Cas12 genome editing, genome integration and CRISPRi implementation. These simple‐to‐use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory. Minimized plasmids and their associated design and experimental protocols have been developed to provide efficient and effective CRISPR‐Cas12 genome editing, genome integration and CRISPRi implementation. These simple‐to‐use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory.

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the ‘evolvability’ of certain enzyme features, thus streamlining and informing protein engineering efforts.

Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1′′-2′) ribose–ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila . In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors. Poly(ADP-ribose) glycohydrolase catabolises poly(ADP-ribose), which is covalently attached to proteins following post-translational modification. In this study, the structure of poly(ADP-ribose) glycohydrolase from Tetrahymena thermophila is reported in complex with the small molecule inhibitor RBPI-3.

Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.

X-ray crystallography and EPR spectroscopy are used to characterize a soluble, oxygen-tolerant reductive dehalogenase from Nitratireductor pacificus pht-3B; the data suggest that the cobalt in the cobalamin cofactor ligates the halogen atom of the substrate, directly abstracting the halogen atom via an oxidative addition. Mechanism of action of a reductive dehalogenase Reductive dehalogenases are cobalamin-dependent enzymes that catalyse the removal of a halogen atom from organohalides, organic molecules that contain one or more halogen atoms. A large proportion of environmental pollutants are organohalides and reductive dehalogenases are responsible for the biological dehalogenation of these compounds, including polychlorinated biphenyls. In this manuscript, the authors used X-ray crystallography and EPR spectroscopy to characterize a soluble, oxygen-tolerant reductive dehalogenase, pht-3B, from Nitratireductor pacificus . Their data suggest that the cobalt in the cobalamin cofactor forms a covalent bond with the halogen atom of the substrate, directly abstracting the halogen atom via an oxidative addition. This mechanism is fundamentally different from the mechanisms of other cobalamin-containing enzymes. These findings will be of relevance to future use of reductive dehalogenases in bioremediation and biocatalysis. Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides 1 . Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle 2 , 3 . Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria 4 , 5 , with substrates including polychlorinated biphenyls or dioxins 6 , 7 . Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies 8 , 9 , 10 , 11 , 12 . Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies 13 , we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

As the vast majority of excavated palaeontological skeletal remains are fragmentary to the extent that they cannot be identified by morphological analysis alone, various molecular methods have been considered to retrieve information from an otherwise underutilised resource. The introduction of collagen fingerprinting, known as Zooarchaeology by Mass Spectrometry (ZooMS), has become one of the most popular approaches to improve taxonomic data yields from fragmentary bone. However, manual laboratory work remains a barrier to the analysis of larger sample numbers. Here we test the incorporation of liquid-handling robots to further develop ZooMS into a more automated technique using samples excavated from Grotte Mandrin, France. By increasing the faunal identifications of the morphological indeterminable remains at layer B2 (~ 42–44 Ka), from 55 to 1215 (1026 of which were processed via AutoZooMS), we identified a wider range of taxa, now including Ursidae and Mammuthus , as well as further hominin remains. AutoZooMS has the capacity to investigate larger proportions of palaeontological assemblages rapidly and cost effectively whilst requiring little human intervention, aiming to improve our understanding of the human past.