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Emergence of antibiotic resistance in pathogenic Mycobacterium tuberculosis (Mtb) has elevated tuberculosis to a serious global threat, necessitating alternate solutions for its eradication. D29 mycobacteriophage can infect and kill several mycobacterial species including Mtb. It encodes an endolysin LysA to hydrolyze host bacteria peptidoglycan for progeny release. We previously showed that out of the two catalytically active domains of LysA [N-terminal domain (NTD) and lysozyme-like domain], NTD, when ectopically expressed in Mycobacterium smegmatis (Msm), is able to kill the bacterium nearly as efficiently as full-length LysA. Here, we dissected the functioning of NTD to develop it as a phage-derived small molecule anti-mycobacterial therapeutic. We performed a large-scale site-directed mutagenesis of the conserved residues in NTD and examined its structure, stability, and function using molecular dynamic simulations coupled with biophysical and biochemical experiments. Our data show that NTD functions as a putative cysteine peptidase with a catalytic triad composed of Cys41, His112, and Glu137, acting as nucleophile, base, and acid, respectively, and showing characteristics similar to the NlpC/P60 family of cysteine peptidases. Additionally, our peptidoglycan hydrolysis assays suggested that NTD hydrolyzes only mycobacterial peptidoglycan and does not act on Gram-positive and Gram-negative bacterial peptidoglycans. More importantly, the combined activity of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills Msm in vitro and exhibits disruption of pre-formed mycobacterial biofilm. We additionally show that NTD treatment increases the permeability of antibiotics in Msm, which reduces the minimum inhibitory concentration of the antibiotics. Collectively, we present NTD as a promising phage-derived therapeutic against mycobacteria.IMPORTANCEMycobacteriophages are the viruses that use mycobacteria as host for their progeny production and, in the process, kill them. Mycobacteriophages are, therefore, considered as promising alternatives to antibiotics for killing pathogenic Mycobacterium tuberculosis. The endolysin LysA produced by mycobacteriophage D29 plays an important role in host cell lysis and virion release. Our work presented here highlights the functioning of LysA's N-terminal catalytic domain (NTD) in order to develop it as phage-derived small molecule therapeutics. We show that combined treatment of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills M. smegmatis, shows synergism by reducing the minimum inhibitory concentration of these antibiotics, and exhibits disruption of pre-formed mature biofilm. These outcomes and our detailed biochemical and biophysical dissection of the protein further pave the way toward engineering and development of NTD as a promising therapeutic against mycobacterial infections such as tuberculosis.

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AIMS: Pancreatic cancer, the 12th-most common cancer, globally, is highly challenging to treat due to its complex epigenetic, metabolic, and genomic characteristics. In pancreatic ductal adenocarcinoma, USP21 acts as an oncogene by stabilizing the long isoform of Transcription Factor 7, thereby activating the Wnt signaling pathway. This study aims to inhibit activation of this pathway through computer-aided drug discovery. Accordingly, four libraries of compounds were designed to target the USP21's catalytic domain (Cys221, His518, Asp534), responsible for its deubiquitinating activity. MAIN METHODS: Utilizing an array of computer-aided drug design methodologies, such as molecular docking, virtual screening, principal component analysis, molecular dynamics simulation, and dynamic cross-correlation matrix, the structural and functional characteristics of the USP21-inhibitor complex were examined. Following the evaluation of the binding affinities, 20 potential ligands were selected, and the best ligand was subjected to additional molecular dynamics simulation study. KEY FINDINGS: The results indicated that the ligand-bound USP21 exhibited reduced structural fluctuations compared to the unbound form, as evident from RMSD, RMSF, Rg, and SASA graphs. ADMET analysis of the top ligand showed promising pharmacokinetic and pharmacodynamic profiles, good bioavailability, and low toxicity. The stable conformations of the proposed drug when bound to their target cavities indicate a robust binding affinity of -9.3 kcal/mol. The drug exhibits an elevated pKi value of 6.82, a noteworthy pIC50 value of 5.972, and a pKd value of 6.023 proving its high affinity and inhibitory potential towards the target. SIGNIFICANCE: In-vitro testing of the top compound (MOLHYB-0436) could lead to its use as a potential treatment for pancreatic cancer.

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The Pseudomonas aeruginosa lipase PaL catalyzes the stereoselective hydrolysis of menthyl propionate to produce L-menthol. The lack of a three-dimensional structure of PaL has so far prevented a detailed understanding of its stereoselective reaction mechanism. Here, the crystal structure of PaL was determined at a resolution of 1.80 Å by single-wavelength anomalous diffraction. In the apo-PaL structure, the catalytic His302 is located in a long loop on the surface that is solvent exposed. His302 is distant from the other two catalytic residues, Asp274 and Ser164. This configuration of catalytic residues is unusual for lipases. Using metadynamics simulations, we observed that the enzyme undergoes a significant conformational change upon ligand binding. We also explored the catalytic and stereoselectivity mechanisms of PaL by all-atom molecular dynamics simulations. These findings could guide the engineering of PaL with an improved diastereoselectivity for L-menthol production.

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DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic α-oxoaldehydes such as methylglyoxal into α-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising α-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro and provide an important clue to the origin and significance of DJ-1 esterase activity.

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SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

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Methylesterases (MESs) hydrolyze carboxylic ester and are important for plant metabolism and defense. However, the understanding of MES' role in strawberries against pathogens remains limited. This study identified 15 FvMESs with a conserved catalytic triad from the Fragaria vesca genome. Spatiotemporal expression data demonstrated the upregulated expression of FvMESs in roots and developing fruits, suggesting growth involvement. The FvMES promoter regions harbored numerous stress-related cis-acting elements and transcription factors associated with plant defense mechanisms. Moreover, FvMES2 exhibited a significant response to Botrytis cinerea stress and showed a remarkable correlation with the salicylic acid (SA) signaling pathway. Molecular docking showed an efficient binding potential between FvMES2 and methyl salicylate (MeSA). The role of FvMES2 in MeSA demethylation to produce SA was further confirmed through in vitro and in vivo assays. After MeSA was applied, the transient overexpression of FvMES2 in strawberries enhanced their resistance to B. cinerea compared to wild-type plants.

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SGNH hydrolase-like fold proteins are serine proteases with the default Asp-His-Ser catalytic triad. Here, we show that these proteins share two unique conserved structural organizations around the active site: (1) the Nuc-Oxy Zone around the catalytic nucleophile and the oxyanion hole, and (2) the Acid-Base Zone around the catalytic acid and base. The Nuc-Oxy Zone consists of 14 amino acids cross-linked with eight conserved intra- and inter-block hydrogen bonds. The Acid-Base Zone is constructed from a single fragment of the polypeptide chain, which incorporates both the catalytic acid and base, and whose N- and C-terminal residues are linked together by a conserved hydrogen bond. The Nuc-Oxy and Acid-Base Zones are connected by an SHLink, a two-bond conserved interaction from amino acids, adjacent to the catalytic nucleophile and base.

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Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic strategy for celiac disease. However, the mechanisms governing the interaction of these enzymes with their substrates remain unclear. In this study, conventional molecular dynamics simulations with a microsecond timescale and targeted molecular dynamics simulations were performed to investigate the native states of mxPEP and scPEP enzymes, as well as their allosteric binding with a representative substrate, namely, Z-Ala-Pro-p-nitroanilide (pNA). The simulations reveal that the native scPEP is in an open state, while the native mxPEP is in a closed state. When pNA approaches a closed mxPEP, it binds to an allosteric pocket located at the first and second ß-sheet of the ß-propeller domain, inducing the opening of this enzyme. Neither enzyme is active in the open or partly-open states. Enzymatic activity is enabled only when the catalytic pocket in the closed state fully accommodates the substrates. The internal capacity of the catalytic pocket of PEP in the closed state determines the maximum size of the gluten peptides that the enzymes can catalyze. The present work provides essential molecular dynamics information for the redesign or engineering of PEP enzymes.

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Methylesterases (MES) are involved in hydrolysis of carboxylic esters, which have substantial roles in plant metabolic activities and defense mechanisms. This study aimed to comprehensively investigate Brassica napus BnMESs and characterize their role in response to Plasmodiophora brassicae stress. Forty-four BnMES members were identified and categorized into three groups based on their phylogenetic relationships and structural similarities. Through functional predictions in the promoter regions and analysis of RNA-Seq data, BnMES emerged as pivotal in growth, development, and stress responses to B. napus, particularly BnMES34, was strongly induced in response to P. brassicae infection. Gene Ontology analyses highlighted BnMES34's role in regulation of plant disease resistance responses. Furthermore, overexpression of BnMES34 in A. thaliana exhibited milder clubroot symptoms, and reduced disease indices, suggesting positive regulatory role of BnMES34 in plant's response to P. brassicae stress. Molecular docking and enzyme activity verification indicated that BnMES34 has the ability to generate salicylic acid via methyl salicylate, and further experimentally validated in vivo. This discovery indicates that the overexpression of BnMES34 in Arabidopsis confers resistance against clubroot disease. Overall, our research suggests that BnMES34 has a beneficial regulatory role in enhancing stress resistance to P. brassicae in B. napus.

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Obesity is a disease of epidemic proportions, with a worrisome upward trend. The high consumption of lipids, a major energy source, leads to obesity because of their high calorific value. Pancreatic lipase (PTL), produced by pancreatic acinar cells, hydrolyzes 50-70% of triacylglycerol (TAG) from food. PTL-related protein 1 (PLRP1) and 2 (PLRP2) are also produced by these cells. In vertebrates, PLRP1 has relatively less lipolytic activity, whereas PLRP2 has an essential role in lipid digestion, especially in infants. In this review, we summarize the structure and function of PTL, PLRP1, and PLRP2, and the metabolic fate of PTL inhibitors. We also discuss the current status of clinical trials on orlistat and its combinations for obesity treatment.

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Through millions of years of the evolutionary journey, contemporary enzymes observed in extant metabolic pathways have evolved to become specialized, in contrast to their ancestors, which displayed promiscuous activities with wider substrate specificities. However, there remain critical gaps in our understanding of how these early enzymes could show such catalytic versatility despite lacking the complex three-dimensional folds of the existing modern-day enzymes. Herein, we report the emergence of a promiscuous catalytic triad by short amyloid peptide based nanofibers that access paracrystalline folds of ß-sheets to expose three residues (lysine, imidazole, and tyrosine) toward solvent. The ordered folded nanostructures could simultaneously catalyze two metabolically relevant chemical transformations via C-O and C-C bond manipulations, displaying both hydrolase and retro-aldolase-like activities. Further, the latent catalytic capabilities of the short peptide based promiscuous folds also helped in processing a cascade transformation, suggesting the important role they might have played in protometabolism and early evolutionary processes.

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Microbial degradation has been confirmed as effective and environmentally friendly approach to remediate phthalates from the environment, and hydrolase is an effective element for contaminant degradation. In the present study, a novel dibutyl phthalate (DBP)-hydrolyzing carboxylesterase (named PS06828) from Pseudomonas sp. PS1 was heterogeneously expressed in E. coli, which was identified as a new member of the lipolytic family VI. Purified PS06828 could efficiently degrade DBP with a wide range of temperature (25-37 °C) and pH (6.5-9.0). Multi-spectroscopy methods combined with molecular docking were employed to study the interaction of PS06828 with DBP. Fluorescence and UV-visible absorption spectra revealed the simultaneous presence of static and dynamic component in the fluorescence quenching of PS06828 by DBP. Synchronous fluorescence and circular dichroism spectra showed inconspicuous alteration in micro-environmental polarity around amino acid residues but obvious increasing of α-helix and reducing of ß-sheet and random coil in protein conformation. Based on the information on exact binding sites of DBP on PS06828 provided by molecular docking, the catalytic mechanism mediated by key residues (Ser113, Asp166, and His197) was proposed and subsequently confirmed by site-directed mutagenesis. The results can strengthen our mechanistic understanding of family VI esterase involved in hydrolysis of phthalic acid esters, and provide a solid foundation for further enzymatic modification.

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Insects have evolved several intricate defense mechanisms to adapt to their chemical environment. Due to their versatile capabilities in hydrolytic biotransformation, insect carboxyl/cholinesterases (CCEs) play vital roles in the development of pesticide resistance, facilitating the adaptation of insects to their host plants, and manipulating insect behaviors through the olfaction system. CCEs confer insecticide resistance through the mechanisms of qualitative or quantitative changes of CCE-mediated enhanced metabolism or target-site insensitivity, and may contribute to the host plant adaptation. CCEs represent the first odorant-degrading enzymes (ODEs) discovered to degrade insect pheromones and plant odors and remain the most promising ODE candidates. Here, we summarize insect CCE classification, currently characterized insect CCE protein structure characteristics, and the dynamic roles of insect CCEs in chemical adaptation.

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The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the ß-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the ß-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations.

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The aim of this work was to map the sequence space of aldoxime dehydratases (Oxds) as enzymes with great potential for nitrile synthesis. Microbes contain an abundance of putative Oxds but fewer than ten Oxds were characterized in total and only two in fungi. In this work, we prepared and characterized a new Oxd (protein gb|EEU37245.1 named OxdFv) from Fusarium vanettenii 77-13-4. OxdFv is distant from the characterized Oxds with a maximum of 36% identity. Moreover, the canonical Oxd catalytic triad RSH is replaced by R141-E187-E303 in OxdFv. R141A and E187A mutants did not show significant activities, but mutant E303A showed a comparable activity as the wild-type enzyme. According to native mass spectrometry, OxdFv contained almost 1 mol of heme per 1 mol of protein, and was composed of approximately 88% monomer (41.8 kDa) and 12% dimer. A major advantage of this enzyme is its considerable activity under aerobic conditions (25.0 ± 4.3 U/mg for E,Z-phenylacetaldoxime at pH 9.0 and 55 °C). Addition of sodium dithionite (reducing agent) and Fe2+ was required for this activity. OxdFv favored (aryl)aliphatic aldoximes over aromatic aldoximes. Substrate docking in the homology model of OxdFv showed a similar substrate specificity. We conclude that OxdFv is the first characterized Oxd of the REE type.

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Cooperative molecular contacts play an important role in protein structure and ligand binding. Here, we constructed a PostgreSQL database that stores structural information in the form of atomic environments and allows flexible mining of molecular contacts. Taking the Ser-His-Asp/Glu catalytic triad as a first test case, we demonstrate that the presence of a carboxylate oxygen atom in the vicinity of a His is associated with shorter Ser-OH..N-His bond in the PDB30 subset. We prospectively mine catalytic triads in unannotated proteins, suggesting catalytic functions for unannotated proteins. As a second test case, we demonstrate that this database system can include ligand atoms, represented by Sybyl atom types, by evaluating the proportion of counter-ions for ligand carboxylate oxygens.

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The 20S proteasome is an attractive drug target for the development of anticancer agents because it plays an important role in cellular protein degradation. It has a threonine residue that can act as a nucleophile to attack inhibitors with an electrophilic warhead, forming a covalent adduct. Fundamental understanding of the reaction mechanism between covalent inhibitors and the proteasome may assist the design and refinement of compounds with the desired activity. In this study, we investigated the covalent inhibition mechanism of an α-keto phenylamide inhibitor of the proteasome. We calculated the noncovalent binding free energy using the PDLD/S-LRA/ß method and the reaction free energy through the empirical valence bond method (EVB). Several possible reaction pathways were explored. Subsequently, we validated the calculated activation and reaction free energies of the most plausible pathways by performing kinetic experiments. Furthermore, the effects of different ionization states of Asp17 on the activation energy at each step were also discussed. The results revealed that the ionization states of Asp17 remarkably affect the activation energies and there is an electrostatic reorganization of Asp17 during the course of the reaction. Our results demonstrate the critical electrostatic effect of Asp17 in the active site of the 20S proteasome.

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Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.

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Chlorophyll degradation plays a myriad of physiological roles in photosynthetic organisms, including acclimation to light environment and nutrient remobilization during senescence. Mg extraction from chlorophyll a is the first and committed step of the chlorophyll degradation pathway. This reaction is catalyzed by the Mg-dechelatase enzyme encoded by Stay-Green (SGR). The reaction mechanism of SGR protein remains elusive since metal ion extraction from organic molecules is not a common enzymatic reaction. Additionally, experimentally derived structural information about SGR or its homologs has not yet been reported. In this study, the crystal structure of the SGR homolog from Anaerolineae bacterium was determined using the molecular replacement method at 1.85 Å resolution. Our previous study showed that three residues-H32, D34, and D62 are essential for the catalytic activity of the enzyme. Biochemical analysis involving mutants of D34 residue further strengthened its importance in the functioning of the dechelatase. Docking simulation also revealed the interaction between the D34 side chain and central Mg ion of chlorophyll a. Structural analysis showed the arrangement of D34/H32/D62 in the form of a catalytic triad that is generally found in hydrolases. The probable reaction mechanism suggests that deprotonated D34 side chain coordinates and destabilizes Mg, resulting in Mg extraction. Besides, H32 possibly acts as a general base catalyst and D62 facilitates H32 to be a better proton acceptor. Taken together, the reaction mechanism of SGR partially mirrors the one observed in hydrolases.

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Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. Thus, it is one of the nonstructural viral proteins essential for the replication. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, it would be possible to use the specific inhibitors of DWV 3Cpro to control DWV infection in honey bees in future. IMPORTANCE The number of managed honey bee (Apis mellifera) colonies has considerably declined in many developed countries in the recent years. Deformed wing virus (DWV) vectored by the mites is the major threat to honey bee colonies and health. To give insight into the mechanism of DWV replication in the host cells, we studied the structure-function relationship of 3C protease (3Cpro), which is necessary to cleave a viral polyprotein at the specific sites to produce the mature proteins. We found that the overall structure, some inhibitors, and processing of 3Cpro are shared between Picornavirales; however, there is diversity in the catalytic triad. DWV 3Cpro is the first viral protease characterized among insect RNA viruses and reveals the evolutionary history of 3Cpro among Picornavirales. Furthermore, DWV 3Cpro inhibitors identified in our study could also be applied to control DWV in honey bees in future.

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