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PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated. Poly-ethylene terephthalate (PET) is a widely used plastic which accumulates in the environment with detrimental consequences. Here the authors report crystal structures of a PET-hydrolyzing enzyme from the microbe Ideonella sakaiensis bound to substrate and product analogs, and suggest a catalytic mechanism for its PET-degrading activity.

Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA–protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer.Topoisomerases have essential roles in transcription, DNA replication, chromatin remodelling and, as recently revealed, 3D genome organization. However, topoisomerases also generate DNA–protein crosslinks coupled with DNA breaks, which are increasingly recognized as a source of disease-causing genomic damage.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation 1 , has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers 2 . Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K—a group of naphthoquinones that includes menaquinone and phylloquinone 3 —confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-4 4 , 5 , was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle 6 . The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis. Biochemical and lipidomic analyses identify an anti-ferroptotic function of vitamin K and reveal ferroptosis suppressor protein 1 (FSP1) as the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle.

Traditional views of cellular metabolism imply that it is passively adapted to meet the demands of the cell. It is becoming increasingly clear, however, that metabolites do more than simply supply the substrates for biological processes; they also provide critical signals, either through effects on metabolic pathways or via modulation of other regulatory proteins. Recent investigation has also uncovered novel roles for several metabolites that expand their signalling influence to processes outside metabolism, including nutrient sensing and storage, embryonic development, cell survival and differentiation, and immune activation and cytokine secretion. Together, these studies suggest that, in contrast to the prevailing notion, the biochemistry of a cell is frequently governed by its underlying metabolism rather than vice versa. This important shift in perspective places common metabolites as key regulators of cell phenotype and behaviour. Yet the signalling metabolites, and the cognate targets and transducers through which they signal, are only beginning to be uncovered. In this Review, we discuss the emerging links between metabolism and cellular behaviour. We hope this will inspire further dissection of the mechanisms through which metabolic pathways and intermediates modulate cell function and will suggest possible drug targets for diseases linked to metabolic deregulation.Metabolites are generally viewed as intermediates or products of metabolism. However, many metabolites are also signalling molecules that regulate metabolic reactions and other processes in development, homeostasis and disease. As such, metabolites can confer adaptive responses to environmental changes.

l -lactate modifies proteins through lactylation 1 , but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular l -lactate sensors required for l -lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to l -lactate with micromolar affinity and they directly catalyse l -lactate for ATP-dependent lactylation on the lysine acceptor end. In response to l -lactate, AARS2 associates with cyclic GMP–AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of l -lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular l -lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS. The tRNA synthases AARS1 and AARS2 are identified as evolutionarily conserved sensors of intracellular l -lactate to mediate the global lysine lactylome.

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage 1 – 4 , and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood 5 – 7 . Although Cas9 variants with greater discrimination against mismatches have been designed 8 – 10 , these suffer from substantially reduced rates of on-target DNA cleavage 5 , 11 . Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants. Cryo-electron microscopy structures of Cas9 during mismatch cleavage provide insight into the mechanisms that control off-target effects of Cas9, which will aid in the future design of high-fidelity Cas9 variants with reduced off-target cleavage.

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene 1 . This approach is fundamentally different from the biosynthesis of short-chain (C 10 –C 25 ) terpenes that are formed from polyisoprenyl diphosphates 2 – 4 . In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature. Chimeric triterpene synthases are identified that catalyse non-squalene-dependent triterpene biosynthesis.

A new engineered version of SpCas9, called HypaCas9, displays enhanced accuracy of editing without significant loss of efficiency at the desired target. Proofreading CRISPR One of the main concerns about the use of CRISPR in genomic editing is the possibility of 'off-target' events. Scientists have been modifying the central enzyme involved in CRISPR editing, Cas9 or its homologues, to reduce this unwanted property. Jennifer Doudna and colleagues describe a new version of this nuclease, HypaCas9, which enables more accurate editing, without substantial loss of efficiency on the desired target. The RNA-guided CRISPR–Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing 1 , 2 , 3 , 4 . High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown 5 , 6 , 7 , 8 , 9 . Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state 10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O-2, is the preferred co-substrate of LPMOs. By controlling H(2)O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O-2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

Emerging artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have long been pursued with sustained efforts. The majority of current candidates have rather poor catalytic activity compared with natural molecules. To tackle this limitation, we design artificial enzymes based on a structurally well-defined Au 25 cluster, namely clusterzymes, which are endowed with intrinsic high catalytic activity and selectivity driven by single-atom substitutions with modulated bond lengths. Au 24 Cu 1 and Au 24 Cd 1 clusterzymes exhibit 137 and 160 times higher antioxidant capacities than natural trolox, respectively. Meanwhile, the clusterzymes demonstrate preferential enzyme-mimicking catalytic activities, with Au 25 , Au 24 Cu 1 and Au 24 Cd 1 displaying compelling selectivity in glutathione peroxidase-like (GPx-like), catalase-like (CAT-like) and superoxide dismutase-like (SOD-like) activities, respectively. Au 24 Cu 1 decreases peroxide in injured brain via catalytic reactions, while Au 24 Cd 1 preferentially uses superoxide and nitrogenous signal molecules as substrates, and significantly decreases inflammation factors, indicative of an important role in mitigating neuroinflammation. Artificial enzymes with reprogrammed and augmented catalytic activity and substrate selectivity have emerged to tackle limitations of noble metals or transition metal oxides. Here, the authors report Au 25 clusterzymes which are endowed with high catalytic activity and selectivity in a range of enzyme-mimicking reactions.