scholarly journals Structural basis of substrate recognition and catalysis by fucosyltransferase 8

2020 ◽  
Vol 295 (19) ◽  
pp. 6677-6688 ◽  
Author(s):  
Michael A. Järvå ◽  
Marija Dramicanin ◽  
James P. Lingford ◽  
Runyu Mao ◽  
Alan John ◽  
...  
Keyword(s):  
Enzyme Catalysis ◽  
Substrate Binding ◽  
Structural Basis ◽  
Simple Modification ◽  
Acceptor Substrate ◽  
The Core ◽  
Donor Substrate ◽  
Substrate Preferences ◽  
Dynamics Simulations ◽  
Core Fucosylation

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

scholarly journals Structural basis of substrate recognition and catalysis by fucosyltransferase 8

2020 ◽  
Author(s):  
Michael A. Järvå ◽  
Marija Dramicanin ◽  
James P. Lingford ◽  
Runyu Mao ◽  
Alan John ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Structural Basis ◽  
Simple Modification ◽  
Acceptor Substrate ◽  
Guanosine Diphosphate ◽  
Antibody Therapeutics ◽  
Donor Substrate ◽  
Substrate Preferences ◽  
Dynamics Simulations ◽  
Core Fucosylation

AbstractFucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique conformational change associated with donor substrate binding; common strategies employed by fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.

scholarly journals Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

2020 ◽  
Vol 477 (15) ◽  
pp. 2791-2805
Author(s):  
Aishat Akere ◽  
Serena H. Chen ◽  
Xiaohan Liu ◽  
Yanger Chen ◽  
Sarath Chandra Dantu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Biochemical Characterization ◽  
Structural Information ◽  
Substrate Recognition ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Donor Substrate ◽  
Substrate Preferences ◽  
Dynamics Simulations ◽  
Experimental Crystal

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

scholarly journals Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillus fumigatus

2020 ◽  
Vol 295 (45) ◽  
pp. 15407-15417
Author(s):  
Daisuke Hira ◽  
Takuya Onoue ◽  
Takuji Oka
Keyword(s):  
Aspergillus Fumigatus ◽  
Structural Model ◽  
Catalytic Domain ◽  
Complex Structure ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Acceptor Substrate ◽  
Substrate Complex ◽  
The Core ◽  
Enzyme Substrate

Fungal cell walls and their biosynthetic enzymes are potential targets for novel antifungal agents. Recently, two mannosyltransferases, namely core-mannan synthases A (CmsA/Ktr4) and B (CmsB/Ktr7), were found to play roles in the core-mannan biosynthesis of fungal-type galactomannan. CmsA/Ktr4 is an α-(1→2)-mannosyltransferase responsible for α-(1→2)-mannan biosynthesis in fungal-type galactomannan, which covers the cell surface of Aspergillus fumigatus. Strains with disrupted cmsA/ktr4 have been shown to exhibit strongly suppressed hyphal elongation and conidiation alongside reduced virulence in a mouse model of invasive aspergillosis, indicating that CmsA/Ktr4 is a potential novel antifungal candidate. In this study we present the 3D structures of the soluble catalytic domain of CmsA/Ktr4, as determined by X-ray crystallography at a resolution of 1.95 Å, as well as the enzyme and Mn2+/GDP complex to 1.90 Å resolution. The CmsA/Ktr4 protein not only contains a highly conserved binding pocket for the donor substrate, GDP-mannose, but also has a unique broad cleft structure formed by its N- and C-terminal regions and is expected to recognize the acceptor substrate, a mannan chain. Based on these crystal structures, we also present a 3D structural model of the enzyme–substrate complex generated using docking and molecular dynamics simulations with α-Man-(1→6)-α-Man-(1→2)-α-Man-OMe as the model structure for the acceptor substrate. This predicted enzyme–substrate complex structure is also supported by findings from single amino acid substitution CmsA/Ktr4 mutants expressed in ΔcmsA/ktr4 A. fumigatus cells. Taken together, these results provide basic information for developing specific α-mannan biosynthesis inhibitors for use as pharmaceuticals and/or pesticides.

scholarly journals Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization

2018 ◽  
Author(s):  
Attabey Rodríguez Benítez ◽  
Sara Tweedy ◽  
Summer A. Baker Dockrey ◽  
April L. Lukowski ◽  
Troy Wymore ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Experimental Data ◽  
Molecular Dynamics ◽  
Protein Engineering ◽  
Molecular Dynamics Simulations ◽  
Substrate Binding ◽  
Structural Basis ◽  
Oxidative Dearomatization ◽  
Dynamics Simulations

Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by the first crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data.

scholarly journals Structural Basis for Selectivity in Flavin-Dependent Monooxygenase-Catalyzed Oxidative Dearomatization

2018 ◽  
Author(s):  
Attabey Rodríguez Benítez ◽  
Sara Tweedy ◽  
Summer A. Baker Dockrey ◽  
April L. Lukowski ◽  
Troy Wymore ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Experimental Data ◽  
Molecular Dynamics ◽  
Protein Engineering ◽  
Molecular Dynamics Simulations ◽  
Substrate Binding ◽  
Structural Basis ◽  
Oxidative Dearomatization ◽  
Dynamics Simulations

Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by the first crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data.

scholarly journals Spatial segregation of mixed-sized counterions in dendritic polyelectrolytes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. S. Kłos ◽  
J. Paturej
Keyword(s):  
Electrostatic Interactions ◽  
Spatial Separation ◽  
Excluded Volume ◽  
The Core ◽  
Bjerrum Length ◽  
Two Component ◽  
Conformational Properties ◽  
Dynamics Simulations ◽  
Two Parameters ◽  
Swelling Factor

AbstractLangevin dynamics simulations are utilized to study the structure of a dendritic polyelectrolyte embedded in two component mixtures comprised of conventional (small) and bulky counterions. We vary two parameters that trigger conformational properties of the dendrimer: the reduced Bjerrum length, $$\lambda _B^*$$ λ B ∗ , which controls the strength of electrostatic interactions and the number fraction of the bulky counterions, $$f_b$$ f b , which impacts on their steric repulsion. We find that the interplay between the electrostatic and the counterion excluded volume interactions affects the swelling behavior of the molecule. As compared to its neutral counterpart, for weak electrostatic couplings the charged dendrimer exists in swollen conformations whose size remains unaffected by $$f_b$$ f b . For intermediate couplings, the absorption of counterions into the pervaded volume of the dendrimer starts to influence its conformation. Here, the swelling factor exhibits a maximum which can be shifted by increasing $$f_b$$ f b . For strong electrostatic couplings the dendrimer deswells correspondingly to $$f_b$$ f b . In this regime a spatial separation of the counterions into core–shell microstructures is observed. The core of the dendrimer cage is preferentially occupied by the conventional ions, whereas its periphery contains the bulky counterions.

scholarly journals Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bertrand Beckert ◽  
Elodie C. Leroy ◽  
Shanmugapriya Sothiselvam ◽  
Lars V. Bock ◽  
Maxim S. Svetlov ◽  
...  
Keyword(s):  
Antibiotic Resistance Genes ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

Insights into the roles of charged residues in substrate binding and mode of action of mannuronan C-5 epimerase AlgE4

Glycobiology ◽  
2021 ◽  
Author(s):  
Margrethe Gaardløs ◽  
Sergey A Samsonov ◽  
Marit Sletmoen ◽  
Maya Hjørnevik ◽  
Gerd Inger Sætrom ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Interdisciplinary Approach ◽  
Product Formation ◽  
Titration Calorimetry ◽  
Binding Groove ◽  
Dynamics Simulations

Abstract Mannuronan C-5 epimerases catalyse the epimerization of monomer residues in the polysaccharide alginate, changing the physical properties of the biopolymer. The enzymes are utilized to tailor alginate to numerous biological functions by alginate-producing organisms. The underlying molecular mechanisms that control the processive movement of the epimerase along the substrate chain is still elusive. To study this, we have used an interdisciplinary approach combining molecular dynamics simulations with experimental methods from mutant studies of AlgE4, where initial epimerase activity and product formation were addressed with NMR spectroscopy, and characteristics of enzyme-substrate interactions were obtained with isothermal titration calorimetry and optical tweezers. Positive charges lining the substrate-binding groove of AlgE4 appear to control the initial binding of poly-mannuronate, and binding also seems to be mediated by both electrostatic and hydrophobic interactions. After the catalytic reaction, negatively charged enzyme residues might facilitate dissociation of alginate from the positive residues, working like electrostatic switches, allowing the substrate to translocate in the binding groove. Molecular simulations show translocation increments of two monosaccharide units before the next productive binding event resulting in MG-block formation, with the epimerase moving with its N-terminus towards the reducing end of the alginate chain. Our results indicate that the charge pair R343-D345 might be directly involved in conformational changes of a loop that can be important for binding and dissociation. The computational and experimental approaches used in this study complement each other, allowing for a better understanding of individual residues’ roles in binding and movement along the alginate chains.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

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