Structural and Biochemical Characterization of PEDV Papain-like Protease 2

2021 ◽  
Author(s):  
Hsu-Feng Chu ◽  
Shu-Chun Cheng ◽  
Chiao-Yin Sun ◽  
Chi-Yuan Chou ◽  
Ta-Hsien Lin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Antiviral Drug ◽  
Substrate Selectivity ◽  
Peptidase Activity ◽  
X Ray ◽  
Diarrhea Virus ◽  
Biochemical Analyses ◽  
Loop 2

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication, but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here we report the crystal structure of the papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that the two residues-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alpha-CoV PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating and proteolytic functions of PEDV PLP2 from a structural perspective. Importance Current challenges in CoVs include a comprehensive understanding of mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structure basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed: (i) three Ub-core interacting residues are essential for DUB function, (ii) two-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only the structural insights to the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

scholarly journals Structural and biochemical characterization of bifunctional XynA

2020 ◽  
Author(s):  
Wei Xie ◽  
Qi Yu ◽  
Yun Liu ◽  
Ruoting Cao ◽  
Ruiqing Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Cellulase Activity ◽  
Cost Savings ◽  
Site Directed Mutagenesis ◽  
Enzyme Activity Assay ◽  
Great Cost ◽  
Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

scholarly journals Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4

2010 ◽  
Vol 55 (3) ◽  
pp. 1248-1255 ◽  
Author(s):  
Patricia Lassaux ◽  
Daouda A. K. Traoré ◽  
Elodie Loisel ◽  
Adrien Favier ◽  
Jean-Denis Docquier ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
Pseudomonas Aeruginosa ◽  
Structural Characterization ◽  
Catalytic Efficiency ◽  
X Ray ◽  
Stabilizing Effect ◽  
Loop 2

ABSTRACTThe metallo-β-lactamase VIM-4, mainly found inPseudomonas aeruginosaorAcinetobacter baumannii, was produced inEscherichia coliand characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

scholarly journals Crystal structure of glycosyltrehalose synthase fromSulfolobus shibataeDSM5389

2018 ◽  
Vol 74 (11) ◽  
pp. 741-746
Author(s):  
Nobuo Okazaki ◽  
Michael Blaber ◽  
Ryota Kuroki ◽  
Taro Tamada
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Hyperthermophilic Archaeon ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sulfolobus Shibatae ◽  
Glucosidic Bond

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeonSulfolobus shibataeDSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the (β/α)8-barrel fold that is conserved as the catalytic domain in the α-amylase family. Three invariant catalytic carboxylic amino acids in the α-amylase family are also found in GTSase at positions Asp241, Glu269 and Asp460 in the catalytic domain. The shape of the catalytic cavity and the pocket size at the bottom of the cavity correspond to the intramolecular transglycosylation mechanism proposed from previous enzymatic studies.

scholarly journals Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 240 ◽  
Author(s):  
Gang Ye ◽  
Xiaowei Wang ◽  
Xiaohan Tong ◽  
Yuejun Shi ◽  
Zhen F. Fu ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Porcine Epidemic Diarrhea Virus ◽  
Catalytic Mechanism ◽  
Antiviral Drug ◽  
Structural Basis ◽  
Transmissible Gastroenteritis Virus ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea ◽  
Transmissible Gastroenteritis ◽  
Viral Polyprotein

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.

Crystal Structure Explains Crystal Habit for the Antiviral Drug Rimantadine Hydrochloride

2014 ◽  
Vol 69 (7) ◽  
pp. 823-828 ◽  
Author(s):  
Anatoly Mishnev ◽  
Dmitrijs Stepanovs
Keyword(s):  
Crystal Structure ◽  
Symmetry Axis ◽  
Antiviral Drug ◽  
Structural Features ◽  
Unit Cell ◽  
Crystal Habit ◽  
Maximal Dimension ◽  
X Ray ◽  
Rimantadine Hydrochloride ◽  
Inner Channel

The crystal structure of the antiviral drug rimantadine hydrochloride, C12H22N+ Cl−, has been elucidated by a single-crystal X-ray structure analysis. The structure consists of 1-(1- adamantyl)ethanamine (rimantadinium) cations and chloride anions. The Cl− anions link the rimantadinium cations via N-H...Cl hydrogen bonds into infinite rectangular chord-like structural units with charged groups in the inner channel and aliphatic groups on the surface, and oriented along the unit cell c axis. In contrast to strong electrostatic and hydrogen bonding inner interactions the chords in the crystal are held together by weak van der Waals forces only. A two-fold symmetry axis passes through the center of the chord. By indexing of the crystal faces it has been shown that the maximal dimension of the needle-like crystals coincides with the direction of the unit cell c axis. These structural features explain the crystal habit and the anisotropy of the mechanical properties of rimantadine hydrochloride crystals observed upon slicing and cleavage.

X-ray Structure of the ZnII β-Lactamase from Bacteroides fragilis in an Orthorhombic Crystal Form

1998 ◽  
Vol 54 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Andrea Carfi ◽  
Emile Duée ◽  
Raquel Paul-Soto ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Bacteroides Fragilis ◽  
Crystal Form ◽  
Water Molecules ◽  
Orthorhombic Crystal ◽  
X Ray ◽  
Class B ◽  
Catalytic Mechanisms ◽  
Zn Ions

β-Lactamases are extracellular or periplasmic bacterial enzymes which confer resistance to β-lactam antibiotics. On the basis of their catalytic mechanisms, they can be divided into two major groups: active-site serine enzymes (classes A, C and D) and the ZnII enzymes (class B). The first crystal structure of a class B enzyme, the metallo-β-lactamase from Bacillus cereus, has been solved at 2.5 Å resolution [Carfi, Pares, Duée, Galleni, Duez, Frère & Dideberg (1995). EMBO J. 14, 4914–4921]. Recently, the crystal structure of the metallo-β-lactamase from Bacteroides fragilis has been determined in a tetragonal space group [Concha, Rasmussen, Bush & Herzberg (1996). Structure, 4, 823–836]. The structure of the metallo-β-lactamase from B. fragilis in an orthorhombic crystal form at 2.0 Å resolution is reported here. The final crystallographic R is 0.196 for all the 32 501 observed reflections in the range 10–2.0 Å. The refined model includes 458 residues, 437 water molecules, four zinc and two sodium ions. These structures are discussed with reference to Zn binding and activity. A catalytic mechanism is proposed which is coherent with metallo-β-lactamases being active with either one Zn ion (as in Aeromonas hydrophila) or two Zn ions (as in B. fragilis) bound to the protein.

scholarly journals Expression, crystallization and preliminary X-ray analysis of McbB, a multifunctional enzyme involved in β-carboline skeleton biosynthesis

2014 ◽  
Vol 70 (10) ◽  
pp. 1402-1405 ◽  
Author(s):  
Hua Wang ◽  
Huaidong Zhang ◽  
Yanling Mi ◽  
Jianhua Ju ◽  
Qi Chen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Data Analysis ◽  
Preliminary Data ◽  
Catalytic Mechanism ◽  
Monoclinic Space Group ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Therapeutic Value ◽  
Ring Structures

β-Carboline alkaloids (βCs), with tricyclic pyrido[3,4-b]indole rings, have important pharmacological and therapeutic value. In the biosynthesis of βCs, the Pictet–Spengler (PS) cyclization reaction is responsible for the formation of ring structures. McbB is one of a few enzymes that are known to catalyse PS cyclization. It can also catalyse decarboxylation and oxidation. Here, the expression, crystallization and preliminary data analysis of McbB are reported. The crystals diffracted to 2.10 Å resolution and belonged to the monoclinic space groupP21, with unit-cell parametersa= 66.06,b= 85.48,c= 106.19 Å, α = 90.00, β = 106.77, γ = 90.00°. These results provide a basis for solving the crystal structure and elucidating the catalytic mechanism for McbB.

scholarly journals Biochemical identification and crystal structure of kynurenine formamidase from Drosophila melanogaster

2012 ◽  
Vol 446 (2) ◽  
pp. 253-260 ◽  
Author(s):  
Qian Han ◽  
Howard Robinson ◽  
Jianyong Li
Keyword(s):  
Crystal Structure ◽  
Drosophila Melanogaster ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Kynurenine Pathway ◽  
Biologically Active ◽  
Protein Architecture ◽  
Fold Family ◽  
Hydrolase Fold ◽  
Biochemical Identification

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the α/β hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.

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