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 class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET‐consuming ...bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended β8‐α6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in‐depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.
Poly(ethylene terephthalate) (PET) plastic material waste causes severe environmental burden worldwide. PET biological decomposition, mediated by a specific enzyme called PETase from a bacterium which can utilize PET as a carbon source, has recently attracted much attention. In this review, the crystal structure of the novel PETase reported from several recent advanced studies is summarized.
Cytochrome P450s are heme‐thiolate enzymes that participate in carbon source assimilation, natural compound biosynthesis and xenobiotic metabolism in all kingdoms of life. P450s can catalyze various ...reactions by using a wide range of organic compounds, thus exhibiting great potential in biotechnological applications. The catalytic reactions of P450s are driven by electron equivalents that are sourced from pyridine nucleotides and delivered by cognate or matching redox partners (RPs). The electron transfer (ET) route from RPs to P450s involves one or more redox center‐containing domains. As the rate of ET is one of the main determinants of P450 efficacy, an in‐depth understanding of the P450 ET pathway should increase our knowledge of these important enzymes and benefit their further applications. Here, the various P450 RP systems along with current understanding of their ET routes will be reviewed. Notably, state‐of‐the‐art structural studies of the two main types of self‐sufficient P450 will also be summarized.
Versatile P450s catalyze various reactions upon acquiring electrons from redox partners. Electrons transferred from the cofactor to heme are of central importance to P450 action, but the route electrons travel remains unveiled. This issue might be probed under the guidance of recent structural studies of self‐sufficient P450s.
Coronaviruses (CoVs) are positive single‐stranded RNA viruses that cause severe respiratory syndromes in humans, including severe acute respiratory syndrome (SARS) and Middle East respiratory ...syndrome (MERS). Coronavirus disease 2019 (COVID‐19) caused by a novel severe acute respiratory syndrome CoV (SARS‐CoV‐2) at the end of 2019 became a global pandemic. The 3C‐like cysteine protease (3CLpro) processes viral polyproteins to yield mature non‐structural proteins, thus playing an important role in the CoV life cycle, and therefore is considered as a prominent target for antiviral drugs. To date, many 3CLpro inhibitors have been reported, and their molecular mechanisms have been illustrated. Here, we briefly introduce the structural features of 3CLpro of the human‐related SARS‐CoV, MERS‐CoV and SARS‐CoV‐2, and explore the potency and mechanism of their cognate inhibitors. This information will shed light on the development and optimization of CoV 3CLpro inhibitors, which may benefit the further designation of therapeutic strategies for treating CoV diseases.
The 3C‐like main protease (3CLpro) of coronaviruses, which causes severe respiratory syndrome in humans, is important for the viral life cycle and is a promising antiviral target. This review summarizes the structural features of 3CLpro from three causative agents of human respiratory infection and compounds that were reported to inhibit protease activity and/or virus replication. This information should help guide drug development against COVID‐19 and other related viruses.
Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, ...triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this “inside-out” triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during “outside” signaling, as measured by single-cell force microscopy. Our findings provide insight into the “inside-out” triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.
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•The crystal structure of HMBPP-bound intracellular BTN3A1 was determined at 1.97 Å•HMBPP forms hydrogen bonds with H 351 for efficient Vγ9Vδ2 T cell activation•An asymmetric intracellular dimer is involved in HMBPP-mediated γδ T cell activation•HMBPP doubles the binding force between extracellular BTN3A and Vγ9Vδ2 TCR
Vγ9Vδ2 T cells sense tumor and microbial metabolites without MHC restriction. Yang et al. used a multifaceted approach to show how the highly potent microbial HMBPP binds to BTN3A1 and triggers inside-out signaling to activate Vγ9Vδ2 T cells. This study will have implications in emerging clinical applications of allogeneic Vγ9Vδ2 T cells.
Polyethylene terephthalate (PET) is among the most extensively produced plastics, but huge amounts of PET wastes that have accumulated in the environment have become a serious threat to the ...ecosystem. Applying PET hydrolytic enzymes to depolymerize PET is an attractive measure to manage PET pollution, and searching for more effective enzymes is a prerequisite to achieve this goal. A thermostable cutinase that originates from the leaf-branch compost termed ICCG is the most effective PET hydrolase reported so far. Here, we illustrated the crystal structure of ICCG in complex with the PET analogue, mono(2-hydroxyethyl)terephthalic acid, to reveal the enzyme–substrate interaction network. Furthermore, we applied structure-based engineering to modify ICCG and screened for variants that exhibit higher efficacy than the parental enzyme. As a result, several variants with the measured melting temperature approaching 99 °C and elevated PET hydrolytic activity were obtained. Finally, crystallographic analyses were performed to reveal the structural stabilization effects mediated by the introduced mutations. These results are of importance in the context of understanding the mechanism of action of the thermostable PET hydrolytic enzyme and shall be beneficial to the development of PET biodegradation platforms.
Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide ...chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the 2Fe-2S cluster. FMN and the 2Fe-2S cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the 2Fe-2S cluster and heme is approx. 25.3 Å. The role of several residues located between the 2Fe-2S cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.
Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated ...globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.
KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS(G12D) variant still lacks inhibitors. Herein, we designed a series of potent inhibitors that can form a ...salt bridge with KRAS's Asp12 residue. Our ITC results show that these inhibitors have similar binding affinity with both GDP-bound and GTP-bound KRAS(G12D), and our crystallographic studies reveal the structural basis of inhibitor binding-induced switch-II pocket in KRAS(G12D), experimentally confirming the formation of a salt bridge between the piperazine moiety of the inhibitors and the Asp12 residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays demonstrate the selectivity of the inhibitors for KRAS(G12D); and the inhibitors disrupt the KRAS-CRAF interaction. We also observed the inhibition of cancer cell proliferation as well as MAPK signaling by a representative inhibitor (TH-Z835). However, since the inhibition was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via targeting non-KRAS small GTPases. Experiments with mouse xenograft models of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a strategy based on salt-bridge and induced-fit pocket formation for KRAS(G12D) targeting, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.
A 2,3‐dihydroxybenzoic acid decarboxylase from Fusarium oxysporum (2,3‐DHBD_Fo) has a relatively high catalytic efficiency for the decarboxylation of 2,3‐dihydroxybenzoic acid (DHBA) and ...carboxylation of catechol, thus it has a different substrate spectrum from other benzoic acid decarboxylases. We have determined the structures of 2,3‐DHBD_Fo in its apo form and complexes with catechol or 2,5‐dihydroxybenzoic acid at 1.55, 1.97, and 2.45 Å resolution, respectively. The crystal structures of 2,3‐DHBD_Fo show that the enzyme exists as a homotetramer, and each active center has a Zn2+ ion coordinated by E8, H167, D291 and three water molecules. This is different from 2,6‐DHBD from Rhizobium sporomusa, in which the Zn2+ ion is also coordinated with H10. Surprisingly, mutation of A10 of 2,3‐DHBD_Fo to His resulted in almost complete loss of the enzyme activity. Enzyme‐substrate docking and site‐directed mutation studies indicate that residue R233Δ interacts with the 3‐hydroxy group of 2,3‐DHBA, and plays an important role in substrate recognition for this enzyme, thus revealing the molecular basis 2,3‐dihydroxybenzoic acid decarboxylase.
It depends on your position: Crystal structure and mutation studies reveal that R233Δ plays an important role in the substrate recognition of 2,3‐dihydroxybenzoic acid decarboxylase (DHBD) from Fusarium oxysporum, as well as the molecular basis for this enzyme being a 2,3‐DHBD with different substrate preference from known 2,6‐DHBDs and salicylic acid decarboxylases.
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