scholarly journals Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Hong-Hsiang Guan ◽  
Yen-Hua Huang ◽  
En-Shyh Lin ◽  
Chun-Jung Chen ◽  
Cheng-Yang Huang
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Structural Similarity ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Molecular Evidence ◽  
Binding Modes ◽  
Pyrimidine Nucleotides ◽  
Quenching Method

Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

scholarly journals Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

2015 ◽  
Vol 81 (12) ◽  
pp. 4216-4223 ◽  
Author(s):  
Mohammad Wadud Bhuiya ◽  
Soon Goo Lee ◽  
Joseph M. Jez ◽  
Oliver Yu
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Ferulic Acid ◽  
Active Site ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Acid Decarboxylase ◽  
Content Type ◽  
Ferulic Acid Decarboxylase

ABSTRACTThe nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) fromSaccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product ofp-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the β2e-α5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ∼30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications.

scholarly journals Crystal structure of papain-E64-c complex. Binding diversity of E64-c to papain S2 and S3 subsites

1992 ◽  
Vol 287 (3) ◽  
pp. 797-803 ◽  
Author(s):  
M J Kim ◽  
D Yamamoto ◽  
K Matsumoto ◽  
M Inoue ◽  
T Ishida ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Cysteine Proteinase ◽  
Complex Structure ◽  
Binding Mode ◽  
Accessible Surface Area ◽  
Cysteine Proteinase Inhibitor ◽  
Binding Modes ◽  
Replacement Method ◽  
Solvent Molecules

In order to investigate the binding mode of E64-c (a synthetic cysteine proteinase inhibitor) to papain at the atomic level, the crystal structure of the complex was analysed by X-ray diffraction at 1.9 A (1 A is expressed in SI units as 0.1 nm) resolution. The crystal has a space group P2(1)2(1)2(1) with a = 43.37, b = 102.34 and c = 49.95 A. A total of 21,135 observed reflections were collected from the same crystal, and 14811 unique reflections of up to 1.9 A resolution [Fo > 3 sigma(Fo)] were used for the structure solution and refinement. The papain structure was determined by means of the molecular replacement method, and then the inhibitor was observed on a (2 magnitude of Fo-magnitude of Fc) difference Fourier map. The complex structure was finally refined to R = 19.4% including 207 solvent molecules. Although this complex crystal (Form II) was polymorphous as compared with the previously analysed one (Form I), the binding modes of leucine and isoamylamide moieties of E64-c were significantly different from each other. By the calculation of accessible surface area for each complex atom, these two different binding modes were both shown to be tight enough to prevent the access of solvent molecules to the papain active site. With respect to the E64-c-papain binding mode, molecular-dynamics simulations proposed two kinds of stationary states which were derived from the crystal structures of Forms I and II. One of these, which corresponds to the binding mode simulated from Form I, was essentially the same as that observed in the crystal structure, and the other was somewhat different from the crystal structure of Form II, especially with respect to the binding of the isoamylamide moiety with the papain S subsites. The substrate specificity for the papain active site is discussed on the basis of the present results.

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

scholarly journals Crystal structure of yeast monothiol glutaredoxin Grx6 in complex with a glutathione-coordinated [2Fe–2S] cluster

2016 ◽  
Vol 72 (10) ◽  
pp. 732-737 ◽  
Author(s):  
Mohnad Abdalla ◽  
Ya-Nan Dai ◽  
Chang-Biao Chi ◽  
Wang Cheng ◽  
Dong-Dong Cao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
Active Site ◽  
Binding Pattern ◽  
Biological Functions ◽  
Structural Comparison ◽  
Multiple Sequence ◽  
Dimeric Structure

Glutaredoxins (Grxs) constitute a superfamily of proteins that perform diverse biological functions. TheSaccharomyces cerevisiaeglutaredoxin Grx6 not only serves as a glutathione (GSH)-dependent oxidoreductase and as a GSH transferase, but also as an essential [2Fe–2S]-binding protein. Here, the dimeric structure of the C-terminal domain of Grx6 (holo Grx6C), bridged by one [2Fe–2S] cluster coordinated by the active-site Cys136 and two external GSH molecules, is reported. Structural comparison combined with multiple-sequence alignment demonstrated that holo Grx6C is similar to the [2Fe–2S] cluster-incorporated dithiol Grxs, which share a highly conserved [2Fe–2S] cluster-binding pattern and dimeric conformation that is distinct from the previously identified [2Fe–2S] cluster-ligated monothiol Grxs.

scholarly journals Expression, crystallization and structure elucidation of γ-terpinene synthase fromThymus vulgaris

2016 ◽  
Vol 72 (1) ◽  
pp. 16-23 ◽  
Author(s):  
Kristin Rudolph ◽  
Christoph Parthier ◽  
Claudia Egerer-Sieber ◽  
Daniel Geiger ◽  
Yves A. Muller ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structure Elucidation ◽  
Structural Similarity ◽  
Enzymatic Conversion ◽  
Terpenoid Biosynthesis ◽  
Mentha Spicata ◽  
Geranyl Diphosphate ◽  
Limonene Synthase ◽  
Putative Active Site

The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil fromThymussp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase fromT. vulgaris(TvTPS), theThymusspecies that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purifiedTvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site ofTvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase fromSalviasp. and to (4S)-limonene synthase fromMentha spicata.

scholarly journals GMP and IMP Are Competitive Inhibitors of CMY-10, an Extended-Spectrum Class C β-Lactamase

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Jung-Hyun Na ◽  
Young Jun An ◽  
Sun-Shin Cha
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Binding Mode ◽  
Competitive Inhibitor ◽  
Phosphate Group ◽  
The Other ◽  
Extended Spectrum ◽  
Competitive Inhibitors ◽  
Class C

ABSTRACT Nucleotides were effective in inhibiting the class C β-lactamase CMY-10. IMP was the most potent competitive inhibitor, with a Ki value of 16.2 μM. The crystal structure of CMY-10 complexed with GMP or IMP revealed that nucleotides fit into the R2 subsite of the active site with a unique vertical binding mode where the phosphate group at one terminus is deeply bound in the subsite and the base at the other terminus faces the solvent.

Fragment Pose Prediction Using Non-Equilibrium Candidate Monte Carlo and Molecular Dynamics Simulations

2019 ◽  
Author(s):  
Nathan M. Lim ◽  
Meghan Osato ◽  
Gregory L. Warren ◽  
David L. Mobley
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Monte Carlo ◽  
Ligand Binding ◽  
Md Simulations ◽  
Binding Mode ◽  
Protein Crystal ◽  
Pose Prediction ◽  
Binding Modes ◽  
Non Equilibrium

<div>Part of early stage drug discovery involves determining how molecules may bind to the target protein. Through understanding where and how molecules bind, chemists can begin to build ideas on how to design improvements to increase binding affinities. In this retrospective study, we compare how computational approaches like docking, molecular dynamics (MD) simulations, and a non-equilibrium candidate Monte Carlo (NCMC) based method (NCMC+MD) perform in predicting binding modes for a set of 12 fragment-like molecules which bind to soluble epoxide hydrolase. We evaluate each method's effectiveness in identifying the dominant binding mode and finding any additional binding modes (if any). Then, we compare our predicted binding modes to experimentally obtained X-ray crystal structures.</div><div>We dock each of the 12 small molecules into the apo-protein crystal structure and then run simulations up to 1 microsecond each. Small and fragment-like molecules likely have smaller energy barriers separating different binding modes by virtue of relatively fewer and weaker interactions relative to drug-like molecules, and thus likely undergo more rapid binding mode transitions. We expect, thus, to see more rapid transitions betweeen binding modes in our study. </div><div><br></div><div>Following this, we build Markov State Models (MSM) to define our stable ligand binding modes. We investigate if adequate sampling of ligand binding modes and transitions between them can occur at the microsecond timescale using traditional MD or a hybrid NCMC+MD simulation approach. Our findings suggest that even with small fragment-like molecules, we fail to sample all the crystallographic binding modes using microsecond MD simulations, but using NCMC+MD we have better success in sampling the crystal structure while obtaining the correct populations.</div>

scholarly journals The C-terminal extension landscape of naturally presented HLA-I ligands

2018 ◽  
Vol 115 (20) ◽  
pp. 5083-5088 ◽  
Author(s):  
Philippe Guillaume ◽  
Sarah Picaud ◽  
Petra Baumgaertner ◽  
Nicole Montandon ◽  
Julien Schmidt ◽  
...  
Keyword(s):  
Crystal Structure ◽  
T Cells ◽  
High Resolution ◽  
Antigen Presentation ◽  
Structural Properties ◽  
Structural Changes ◽  
Binding Mode ◽  
Binding Modes ◽  
The Common ◽  
Direct Recognition

HLA-I molecules play a central role in antigen presentation. They typically bind 9- to 12-mer peptides, and their canonical binding mode involves anchor residues at the second and last positions of their ligands. To investigate potential noncanonical binding modes, we collected in-depth and accurate HLA peptidomics datasets covering 54 HLA-I alleles and developed algorithms to analyze these data. Our results reveal frequent (442 unique peptides) and statistically significant C-terminal extensions for at least eight alleles, including the common HLA-A03:01, HLA-A31:01, and HLA-A68:01. High resolution crystal structure of HLA-A68:01 with such a ligand uncovers structural changes taking place to accommodate C-terminal extensions and helps unraveling sequence and structural properties predictive of the presence of these extensions. Scanning viral proteomes with the C-terminal extension motifs identifies many putative epitopes and we demonstrate direct recognition by human CD8+ T cells of a 10-mer epitope from cytomegalovirus predicted to follow the C-terminal extension binding mode.

scholarly journals Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Markus A Jobst ◽  
Lukas F Milles ◽  
Constantin Schoeler ◽  
Wolfgang Ott ◽  
Daniel B Fried ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Conformation ◽  
Bacterial Species ◽  
Force Spectroscopy ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Binding Modes ◽  
Single Molecule Force Spectroscopy ◽  
Vast Number ◽  
Knock Out

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicteddual binding modes across multiple bacterial species, our approach opens up newpossibilities for understanding assembly and catalytic properties of a broadrange of multi-enzyme complexes.

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