Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum

Abstract

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose–methanol–choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His–Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da.

Development of a Crystallographic Screening to Identify Sudan Virus VP40 Ligands

“…We successfully employed fragment-based lead design via crystal soaking and identified salicylic acid as a crystallographic binder of dimeric sVP40. The success rate of the screening described here was surprisingly low, with only one fragment identified as a hit when compared to other publications using the same library. ,, Other fragments of the Jena FragXtal screen that are structurally quite similar to SA did not bind, suggesting that the functional groups are crucial for successful soaking (Table S1). Due to its small size, SA seems to be a promiscuous binder, as it was reported as a ligand for numerous other proteins, − also including screenings using the same library. , SA derivatives such as 2-hydroxy-4-aminobenzoic acid and P -hydroxybenzoic acid are bound to the SARS-CoV-2 NSP3 macrodomain in the crystalline state (PDB-codes: 5RUE and 5RTJ, respectively) …”

“…For Astex Ro3, fragments obey limitations such as molecular weight ≤300 Da and number of hydrogen bond donors and acceptors ≤3 . This library was already used for a number of screening analyses. ,,, All fragments were dissolved in dimethylsulfoxide (DMSO) and diluted in crystallization buffer to 100 mM (final concentration of 10% v/v). One or several crystals were then soaked with each fragment for 12–16 h, 1 h or 10 s, flash-frozen, and analyzed via X-ray diffraction.…”

“…The success rate of the screening described here was surprisingly low, with only one fragment identified as a hit when compared to other publications using the same library. 28 , 34 , 42 Other fragments of the Jena FragXtal screen that are structurally quite similar to SA did not bind, suggesting that the functional groups are crucial for successful soaking ( Table S1 ). Due to its small size, SA seems to be a promiscuous binder, as it was reported as a ligand for numerous other proteins, 43 − 52 also including screenings using the same library.…”

Comparison of two peroxidases with high potential for biotechnology applications – HRP vs. APEX2

“…Starting from the available crystal structures of APX – Ascorbate peroxidase from soybean cytosol of Glycine max (PDB code 1OAG) [4] and HRP – Horseradish peroxidase C1A from Armoracia rusticana (PDB code 1H5A) [8] , four systems were prepared for molecular dynamics simulations: wild type (i) APX and (ii) HRP, and engineered split forms (iii) sAPEX2 and (iv) sHRP. ABTS coordinates were taken from crystal structure complex with FAD-dependent oxidoreductase (PDB code 7AA2) [37] . sHRP was prepared from HRP structure by introducing mutations of 6 amino acids (T21I, P78S, R93G, N175S, N255D, L299R) and split between residues 213–214 [16] .…”

“…Docking study was performed using and ensemble of 10 structures of HRP protein gathered from the 500 ns long MD simulation at fixed time intervals, as well as on the equilibrated structure of HRP protein (structure obtained after geometry optimization and short 10 ns equilibration). The structure of the ligand ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was directly taken from the protein data bank (PDB code 7AA2, crystal structure of ABTS in complex with oxidoreductase) [37] . Protein and ligand parametrization was performed with AutoDock Tools4 [57] using the AutoDock 4.2 atom typing.…”