Biological Activity of Insecticidal Toxins: Structural Basis, Site-Directed Mutagenesis and Perspectives

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

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Structural basis for the functional properties of the P2X7 receptor for extracellular ATP

Purinergic Signalling ◽

10.1007/s11302-021-09790-x ◽

2021 ◽

Author(s):

Lin-Hua Jiang ◽

Emily A. Caseley ◽

Steve P. Muench ◽

Sébastien Roger

Keyword(s):

Functional Properties ◽

P2x7 Receptor ◽

Site Directed Mutagenesis ◽

P2x Receptor ◽

Structural Basis ◽

Structure Characteristic ◽

Purinergic Signalling ◽

Directed Mutagenesis ◽

Important Mediator ◽

Receptor Family

AbstractThe P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure–function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor.

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Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing.

Molecular and Cellular Biology ◽

10.1128/mcb.6.4.1343 ◽

1986 ◽

Vol 6 (4) ◽

pp. 1343-1348 ◽

Cited By ~ 10

Author(s):

M Hannink ◽

D J Donoghue

Keyword(s):

Biological Activity ◽

Growth Factor ◽

Gene Product ◽

Signal Sequence ◽

Intracellular Localization ◽

Growth Factor Receptor ◽

Proteolytic Processing ◽

Site Directed Mutagenesis ◽

Platelet Derived Growth Factor ◽

Directed Mutagenesis

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.

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Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

Chemical Senses ◽

10.1093/chemse/bjz015 ◽

2019 ◽

Vol 44 (5) ◽

pp. 303-310 ◽

Cited By ~ 1

Author(s):

Jean-Baptiste Chéron ◽

Amanda Soohoo ◽

Yi Wang ◽

Jérôme Golebiowski ◽

Serge Antonczak ◽

...

Keyword(s):

Transmembrane Domain ◽

Taste Receptor ◽

Receptor Activation ◽

Sweet Taste ◽

Site Directed Mutagenesis ◽

Activation Mechanism ◽

Structural Basis ◽

Directed Mutagenesis ◽

Conserved Residues ◽

Sweet Taste Receptor

Abstract Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

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Structure and allosteric regulation of eukaryotic 6-phosphofructokinases

Biological Chemistry ◽

10.1515/hsz-2013-0130 ◽

2013 ◽

Vol 394 (8) ◽

pp. 977-993 ◽

Cited By ~ 30

Author(s):

Torsten Schöneberg ◽

Marco Kloos ◽

Antje Brüser ◽

Jürgen Kirchberger ◽

Norbert Sträter

Keyword(s):

Crystal Structures ◽

Structural Information ◽

Current Knowledge ◽

Allosteric Regulation ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Directed Mutagenesis ◽

Special Focus ◽

Molecular Architecture ◽

Key Enzyme

Abstract Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.

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Structural Basis of Hematopoietic Prostaglandin D Synthase Activity Elucidated by Site-directed Mutagenesis

Journal of Biological Chemistry ◽

10.1074/jbc.m000750200 ◽

2000 ◽

Vol 275 (40) ◽

pp. 31239-31244 ◽

Cited By ~ 47

Author(s):

Elena Pinzar ◽

Masashi Miyano ◽

Yoshihide Kanaoka ◽

Yoshihiro Urade ◽

Osamu Hayaishi

Keyword(s):

Site Directed Mutagenesis ◽

Structural Basis ◽

Directed Mutagenesis ◽

Prostaglandin D Synthase

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Identification of Critical Residues for Ligand Binding in the Integrin β3 I-domain by Site-directed Mutagenesis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613076 ◽

2002 ◽

Vol 87 (04) ◽

pp. 756-762 ◽

Cited By ~ 10

Author(s):

Jun Yamanouchi ◽

Tatsushiro Tamura ◽

Shigeru Fujita ◽

Takaaki Hato

Keyword(s):

Ligand Binding ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Ligand Recognition ◽

Directed Mutagenesis ◽

Different Types ◽

Β3 Integrin ◽

Critical Residues ◽

Α Subunits ◽

Putative Ligand Binding

SummaryTo define the structural basis of ligand recognition by αIIb β3, we conducted site-directed mutagenesis of residues located on the top surface of the β3 I-domain that is homologous to the I-domain of several α subunits and contains a putative ligand binding site. Here we identify D158 and N215 in β3 as novel residues critical for ligand binding. Alanine substitution of D158 or N215 abolished binding of a ligand-mimetic antibody and fibrinogen to αIIb β3 induced by different types of integrin activation. CHO cells expressing recombinant αIIb β3 bearing D158A or N215A mutation did not adhere to fibrinogen. These mutations had the same effect on ligand binding to another β3 integrin, αV β3. Compared to the αI-domain structure, the βB-βC loop containing D158 in the β3 I-domain is quite different in length and sequence. These results suggest that the structure for ligand recognition is different in the βI- and αI-domains.

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Structural Basis of Binding of High-Affinity Ligands to Protein Kinase C: Prediction of the Binding Modes through a New Molecular Dynamics Method and Evaluation by Site-Directed Mutagenesis

Journal of Medicinal Chemistry ◽

10.1021/jm000488e ◽

2001 ◽

Vol 44 (11) ◽

pp. 1690-1701 ◽

Cited By ~ 31

Author(s):

Youngshang Pak ◽

Istvan J. Enyedy ◽

Judith Varady ◽

Justin W. Kung ◽

Patricia S. Lorenzo ◽

...

Keyword(s):

Molecular Dynamics ◽

Protein Kinase C ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Directed Mutagenesis ◽

Binding Modes ◽

Affinity Ligands ◽

High Affinity ◽

Kinase C ◽

Dynamics Method

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Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.3777-3783.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 3777-3783 ◽

Cited By ~ 61

Author(s):

Christiane Szekat ◽

Ralph W. Jack ◽

Dirk Skutlarek ◽

Harald Färber ◽

Gabriele Bierbaum

Keyword(s):

Biological Activity ◽

Expression System ◽

Biosynthetic Gene Cluster ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Lipid Ii ◽

Type Gene ◽

Self Protection ◽

Acid Function ◽

Reduced Activity

ABSTRACT The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.

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