Structures of the methyltransferase component ofDesulfitobacterium hafnienseDCB-2O-demethylase shed light on methyltetrahydrofolate formation

2015 ◽  
Vol 71 (9) ◽  
pp. 1900-1908 ◽  
Author(s):  
Hanno Sjuts ◽  
Mark S. Dunstan ◽  
Karl Fisher ◽  
David Leys
Keyword(s):  
Proton Transfer ◽  
Crystal Structures ◽  
Active Site ◽  
Active Site Residues ◽  
Methyl Group ◽  
Enzyme Systems ◽  
Desulfitobacterium Hafniense ◽  
Shed Light

O-Demethylation by acetogenic or organohalide-respiring bacteria leads to the formation of methyltetrahydrofolate from aromatic methyl ethers.O-Demethylases, which are cobalamin-dependent, three-component enzyme systems, catalyse methyl-group transfers from aromatic methyl ethers to tetrahydrofolateviamethylcobalamin intermediates. In this study, crystal structures of the tetrahydrofolate-binding methyltransferase module from aDesulfitobacterium hafnienseDCB-2O-demethylase were determined both in complex with tetrahydrofolate and the product methyltetrahydrofolate. While these structures are similar to previously determined methyltransferase structures, the position of key active-site residues is subtly altered. A strictly conserved Asn is displaced to establish a putative proton-transfer network between the substrate N5 and solvent. It is proposed that this supports the efficient catalysis of methyltetrahydrofolate formation, which is necessary for efficientO-demethylation.

scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (7) ◽  
pp. 1607-1616 ◽  
Author(s):  
Ji-Hye Lee ◽  
Intekhab Alam ◽  
Kang Rok Han ◽  
Sunyoung Cho ◽  
Sungho Shin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Health Concern ◽  
Murine Norovirus ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Close Proximity ◽  
Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals Biological and Structural Characterization of Trypanosoma cruzi Phosphodiesterase C and Implications for Design of Parasite Selective Inhibitors

2012 ◽  
Vol 287 (15) ◽  
pp. 11788-11797 ◽  
Author(s):  
Huanchen Wang ◽  
Stefan Kunz ◽  
Gong Chen ◽  
Thomas Seebeck ◽  
Yiqian Wan ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Trypanosoma Evansi ◽  
Active Site Residues ◽  
Dual Specificity ◽  
Pde Inhibitors ◽  
Starting Model

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a Km of 23.8 μm and a kcat of 31 s−1 for cAMP and a Km of 99.1 μm and a kcat of 17 s−1 for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC50 of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.

scholarly journals Enantioseparation, in vitro testing, and structural characterization of triple-binding reactivators of organophosphate-inhibited cholinesterases

2020 ◽  
Vol 477 (15) ◽  
pp. 2771-2790 ◽  
Author(s):  
Nikola Maraković ◽  
Anamarija Knežević ◽  
Igor Rončević ◽  
Xavier Brazzolotto ◽  
Zrinka Kovarik ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Structural Characterization ◽  
Active Site ◽  
In Vitro Testing ◽  
Active Site Residues

The enantiomers of racemic 2-hydroxyimino-N-(azidophenylpropyl)acetamide-derived triple-binding oxime reactivators were separated, and tested for inhibition and reactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibited with tabun (GA), cyclosarin (GF), sarin (GB), and VX. Both enzymes showed the greatest affinity toward the methylimidazole derivative (III) of 2-hydroxyimino-N-(azidophenylpropyl)acetamide (I). The crystal structure was determined for the complex of oxime III within human BChE, confirming that all three binding groups interacted with active site residues. In the case of BChE inhibited by GF, oximes I (kr = 207 M−1 min−1) and III (kr = 213 M−1 min−1) showed better reactivation efficiency than the reference oxime 2-PAM. Finally, the key mechanistic steps in the reactivation of GF-inhibited BChE with oxime III were modeled using the PM7R6 method, stressing the importance of proton transfer from Nε of His438 to Oγ of Ser203 for achieving successful reactivation.

scholarly journals Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease

2018 ◽  
Author(s):  
Ayşegül Özen ◽  
Kuan-Hung Lin ◽  
Keith P Romano ◽  
Davide Tavella ◽  
Alicia Newton ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Md Simulations ◽  
Drug Susceptibility ◽  
Active Site Residues ◽  
Allosteric Coupling ◽  
Direct Acting ◽  
Distal Mutation ◽  
Active Site Mutations

AbstractHepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 Å away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the α-2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site.

scholarly journals Dual-Function Enzyme Catalysis for Enantioselective Carbon–Nitrogen Bond Formation

2021 ◽  
Author(s):  
zhen liu ◽  
Carla Calvó-Tusell ◽  
Andrew Z. Zhou ◽  
kai chen ◽  
Marc Garcia-Borràs ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Active Site ◽  
Density Functional ◽  
Dual Function ◽  
Cytochrome P450 Enzymes ◽  
Active Site Residues ◽  
Bond Forming ◽  
Enzymatic Transformation ◽  
Excellent Activity ◽  
Dynamics Simulations

<p>Whereas enzymatic asymmetric carbene N–H insertion is a powerful method for preparation of chiral amines in principle, it has suffered from limited enantioselectivity in practice. In this work, we demonstrate that engineered cytochrome P450 enzymes can catalyze this abiological C–N bond-forming reaction with excellent activity and selectivity (up to 32,100 TTN, >99% yield and 98% e.e.) to prepare a series of bioactive <i>α</i>-amino lactones, which have not been accessed previously using a carbene insertion strategy. The enzymes are dual-function catalysts, effecting both carbene transfer and enantioselective proton-transfer catalysis, in a single active site. To gain insight into the mechanism of the enzymatic transformation, especially in the asymmetric protonation step, we performed extensive molecular dynamics simulations and density functional theory (DFT) calculations. Computational studies uncover the important roles of active-site residues that enable high activity and selectivity through interacting with the carbene intermediate and the amine substrate, and directing water molecules for selective proton transfer.<br></p><p></p>

scholarly journals Dual-Function Enzyme Catalysis for Enantioselective Carbon–Nitrogen Bond Formation

2021 ◽  
Author(s):  
zhen liu ◽  
Carla Calvó-Tusell ◽  
Andrew Z. Zhou ◽  
kai chen ◽  
Marc Garcia-Borràs ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Active Site ◽  
Density Functional ◽  
Dual Function ◽  
Cytochrome P450 Enzymes ◽  
Active Site Residues ◽  
Bond Forming ◽  
Enzymatic Transformation ◽  
Excellent Activity ◽  
Dynamics Simulations

<p>Whereas enzymatic asymmetric carbene N–H insertion is a powerful method for preparation of chiral amines in principle, it has suffered from limited enantioselectivity in practice. In this work, we demonstrate that engineered cytochrome P450 enzymes can catalyze this abiological C–N bond-forming reaction with excellent activity and selectivity (up to 32,100 TTN, >99% yield and 98% e.e.) to prepare a series of bioactive <i>α</i>-amino lactones, which have not been accessed previously using a carbene insertion strategy. The enzymes are dual-function catalysts, effecting both carbene transfer and enantioselective proton-transfer catalysis, in a single active site. To gain insight into the mechanism of the enzymatic transformation, especially in the asymmetric protonation step, we performed extensive molecular dynamics simulations and density functional theory (DFT) calculations. Computational studies uncover the important roles of active-site residues that enable high activity and selectivity through interacting with the carbene intermediate and the amine substrate, and directing water molecules for selective proton transfer.<br></p><p></p>

scholarly journals The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

2018 ◽  
Vol 151 (3) ◽  
pp. 282-291
Author(s):  
Kristen A. Gaffney ◽  
Heedeok Hong
Keyword(s):  
Hydrogen Bonding ◽  
Crystal Structures ◽  
Weak Interaction ◽  
Active Site ◽  
Critical Role ◽  
Interaction Energies ◽  
Active Site Residues ◽  
Rhomboid Protease ◽  
Rhomboid Proteases ◽  
Catalytic Dyad

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔGInter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

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