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

Leona Švecová,David Sedlák,Jindřich Hašek,Kirk Matthew Schnorr,Tereza Skálová,Jarmila Dušková,Petr Kolenko,Tomáš Koval',Mária Trundová,Lars Henrik Østergaard,Jan Stránský,Jan Dohnálek

doi:10.1107/s2059798321003533

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.

Highlights

The thermophilic filamentous fungus Chaetomium thermophilum (Ct) found in soil, dung and compost heaps significantly participates in cellulose decomposition
The enzyme was successfully expressed in A. oryzae and the resulting product was purified to homogeneity
The identity of CtFDO was verified by LC-MS/MS spectrometry, which confirmed 595 amino-acid residues and revealed 20 missing residues at the N-terminus (21 residues of the signal peptide are not present) and eight residues at the C-terminus compared with the expected mature enzyme sequence (Supplementary Fig. Subsite 2 (S2))

Summary

Introduction

The thermophilic filamentous fungus Chaetomium thermophilum (Ct) found in soil, dung and compost heaps significantly participates in cellulose decomposition. The conserved histidine plays the role of the catalytic base in the majority of GMC oxidoreductases (Romero & Gadda, 2014; Wohlfahrt et al, 1999; Hernandez-Ortega et al, 2012; Yoshida et al, 2015; Wongnate & Chaiyen, 2013; Mugo et al, 2013; Graf et al, 2015; Smitherman et al, 2015; Dijkman et al, 2015) and is usually present in combination with another histidine or an asparagine residue, which can form a hydrogen bond to an alcohol substrate Both residues are situated on the re face of the FAD isoalloxazine ring, creating a His–His or His–Asn pair (Sutzl et al, 2019)

Methods

Results

Discussion

Conclusion