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Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity 1 – 3 . In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues 4 . Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism 5 , 6 , but these proteins do not mediate Ubl–target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1–E2–Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria. A study reports the structure and molecular mechanism of the Bil anti-phage defence system, demonstrating that it is the closest prokaryotic homologue of canonical eukaryotic ubiquitination pathways.

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state. The molecular mechanism of ubiquitin transfer from E1 to E2 enzymes is still unclear. By solving the crystal structure of a covalently trapped E1–E2–ubiquitin thioester mimetic, the authors identify two conformations of this complex which suggest an affinity switch mechanism for thioester transfer.

Ubiquitin (Ub) signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3. Cdc34 is an E2 that plays a key role in regulating cell cycle progression and requires unique structural elements to function. The molecular basis by which Cdc34 engages its E1 and the structural mechanisms by which its unique C-terminal extension functions in Cdc34 activity are unknown. Here, we present crystal structures of Cdc34 alone and in complex with E1, and a Cdc34~Ub thioester mimetic that represents the product of Uba1-Cdc34 Ub transthiolation. These structures reveal conformational changes in Uba1 and Cdc34 and a unique binding mode that are required for transthiolation. The Cdc34~Ub structure reveals contacts between the Cdc34 C-terminal extension and Ub that stabilize Cdc34~Ub in a closed conformation and are critical for Ub discharge. Altogether, our structural, biochemical, and cell-based studies provide insights into the molecular mechanisms by which Cdc34 function in cells. The E2 enzyme Cdc34 plays a critical role in cell cycle progression but the structural bases for its activities are unknown. Here, the authors present crystal structures of Cdc34 alone, in complex with E1, and in complex with Ub that provide insights into the mechanism of Cdc34 activity in the cell.

Cross-linked enzyme aggregates (CLEA) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents, and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms product per kilogram biocatalyst) and are easy to recover and recycle. Yet another advantage derives from the possibility to co-immobilize two or more enzymes to provide CLEAs that are capable of catalyzing multiple biotransformations, independently or in sequence as catalytic cascade processes. [PUBLICATION ABSTRACT]

The pulp and paper industry discharges massive amount of wastewater containing hazardous organochlorine compounds released during different processing stages. Therefore, some cost-effective and nonpolluting practices such as enzymatic treatments are required for the potential mitigation of effluents released in the environment. Various xylanolytic enzymes such as xylanases, laccases, cellulases and hemicellulases are used to hydrolyse raw materials in the paper manufacturing industry. These enzymes are used either individually or in combination, which has the efficient potential to be considered for bio-deinking and bio-bleaching components. They are highly dynamic, renewable, and high in specificity for enhancing paper quality. The xylanase act on the xylan and cellulases act on the cellulose fibers, and thus increase the bleaching efficacy of paper. Similarly, hemicellulase enzyme like endo-xylanases, arabinofuranosidase and β-d-xylosidases have been described as functional properties towards the biodegradation of biomass. In contrast, laccase enzymes act as multi-copper oxidoreductases, bleaching the paper by the oxidation and reduction process. Laccases possess low redox potential compared to other enzymes, which need some redox mediators to catalyze. The enzymatic process can be affected by various factors such as pH, temperature, metal ions, incubation periods, etc. These factors can either increase or decrease the efficiency of the enzymes. This review draws attention to the xylanolytic enzyme-based advanced technologies for pulp bleaching in the paper industry.

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in the first step of Ub/Ubl conjugation cascades and represent potential targets for therapeutic intervention in cancer and other life-threatening diseases. Here, we report the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a recently discovered and highly specific covalent allosteric inhibitor (COH000). The structure reveals that COH000 targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that COH000 binding to SUMO E1 is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation. These structural changes include disassembly of the active site and a 180° rotation of the catalytic cysteine-containing SCCH domain, relative to conformational snapshots of SUMO E1 poised to catalyze adenylation. Altogether, our study provides a molecular basis for the inhibitory mechanism of COH000 and its SUMO E1 specificity, and also establishes a framework for potential development of molecules targeting E1 enzymes for other Ubls at a cryptic allosteric site. Ubiquitin and ubiquitin-like modifiers such as SUMO play important roles in several cellular pathways that can become deregulated in cancer. Here the authors describe the structural basis for inhibition of SUMO E1 ligase by the small molecule COH000.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal. In order to find a general treatment for cancer, this study found that MTH1 activity is essential for the survival of transformed cells, and isolated two small-molecule inhibitors of MTH1, TH287 and TH588 — in the presence of these inhibitors, damaged nucleotides are incorporated into DNA only in cancer cells, causing cytotoxicity and eliciting a beneficial response in patient-derived mouse xenograft models. MTH1 is Ras-linked target for cancer therapy Mutations in the Ras oncogene are associated with poor prognosis. It was known that overexpression of MTH1, a protein involved in preventing the incorporation of damaged bases into DNA, prevents Ras-induced senescence. In seeking to understand how damaged deoxynucleotides (dNTPs) promote cancer, Thomas Helleday and colleagues found that MTH1 activity is essential for the survival of transformed cells, and isolated two small-molecule MTH1 inhibitors, TH287 and TH588. In the presence of these hydrolase inhibitors, damaged nucleotides are incorporated into DNA only in cancer cells, causing cytotoxicity and eliciting a beneficial response in mouse xenograft cancer models. In a second study, Giulio Superti-Furga and colleagues sought to identify the target of a small molecule, SCH51344, that had been developed for use against Ras -dependent cancers and found that it inactivates MTH1. This allowed them to identify a new potent inhibitor of MTH1 that is enantiomer-selective, ( S )-crizotinib. In the presence of this drug, tumour growth is suppressed in animal models of colon cancer.

Cistus genus (Cistaceae) comprises several medicinal plants used in traditional medicines to treat several pathological conditions including hyperglycemia. These include Cistus salviifolius L. (CS) and Cistus monspeliensis L. (CM), still not fully explored as a source of metabolites with therapeutic potential for human diseases. In this study, the antioxidant α-amylase and α-glucosidase enzyme inhibitory effects of aqueous and hydromethanolic extracts from the aerial parts of Moroccan CS and CM were investigated. Antioxidant activity has been assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and ferric reducing/antioxidant power (FRAP) methods. The α-amylase and α-glucosidase inhibitory activity has been assessed using an in vitro model. Moreover, mineral and phenolic contents of CS and CM were analyzed. The extracts of both species exhibited potent antioxidant activity in all used systems and possess strong inhibitory effect towards α-glucosidase (IC50: 0.95 ± 0.14 to 14.58 ± 1.26 μg/mL) and significant inhibitory potential against α-amylase (IC50: 217.10 ± 0.15 to 886.10 ± 0.10 μg/mL). Furthermore, the result showed high levels of phenolic content and unexpectedly some higher levels of mineral content in CS. The results suggest that the phenolic rich extracts of CS and CM may have a therapeutic potential against diseases associated with oxidative stress and may be useful in the management of hyperglycemia in diabetic patients.

Enzyme-powered nanomotors have attracted significant attention in materials science and biomedicine for their biocompatibility, versatility, and the use of biofuels in biological environments. Here, we employ a hybrid mesoscale method combining molecular dynamics and multi-particle collision dynamics (MD–MPC) to study the dynamics of nanomotors powered by enzyme reactions. Two cascade enzymes are constructed to be layered on the same surface of a Janus colloid, providing a confined space that greatly enhances reaction efficiency. Simulations indicate that such a configuration significantly improves the utilization of intermediate products and, consequently, increases the self-propulsion of the Janus motor. By presenting the gradient fields of substrates and products, as well as the hydrodynamics surrounding the motor, we explore the underlying mechanism behind the enhanced autonomous velocity. Additionally, we discuss the improvements in environmental safety of the modified motor, which may shed light on the fabrication of biocatalytic nano-machines in experiments.

Photoregulation of biomolecules has become crucial tools in chemical biology, because light enables access under mild conditions and with delicate spatiotemporal control. The control of enzyme activity in a reversible way is a challenge. To achieve it, a facile approach is to use photoswitchable inhibitors. This review highlights recent progress in photoswitchable inhibitors based on azobenzenes units. The progress suggests that the incorporation of an azobenzene unit to a known inhibitor is an effective method for preparing a photoswitchable inhibitor, and with these photoswitchable inhibitors, the activity of enzymes can be regulated by optical control, which is valuable in both basic science and therapeutic applications.