Structural data-based identification of substrate nature for novel FAD-dependent oxidoreductase from Chaetomium thermophilum

L. Švecová^{1, 2}, T. Skálová¹, T. Kovaľ¹, L. H. Østergaard³, J. Dohnálek¹

¹Institute of Biotechnology of the Czech Academy of Sciences, v.v.i., Biocev center, Průmyslová 595, Vestec, 252 50, Czech Republic

²Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Břehová 7,115 19, Praha 1, Czech Republic

³Novozymes A/S, Brudelysvej 26, DK-2880 Bagsværd, Denmark

leona.svecova@ibt.cas.cz

Chaetomium thermophilum is a thermophilic cellulose-degrading fungus living in soil, dung, and compost heaps. It flourishes at higher temperatures (between 45 – 60 °C) and, on that account, it is of wide interest as potential source of thermostable enzymes for high-temperature industrial processes [1]. The subject of our study is a novel thermostable FAD-dependent oxidoreductase from Chaetomium thermophilum var. thermophilum (CtFDO), which is an extracellular glycoprotein of molecular mass around 85 kDa.

Here we present a 1.3 Å resolution crystal structure of CtFDO. It belongs to the glucose-methanol-choline (GMC) oxidoreductase family, members of which share the two-domain character, core structural elements, the conserved N-terminal GxGxxG sequence motif characteristic for the Rossmann fold binding the ADP moiety of flavine adenine dinucleotide, and a conserved active-site histidine [2]. A usual feature is also a narrow tunnel or a cleft to the active site pocket containing typically His–His, or His–Asn active-site pair in the re-face of FAD isoalloxazine ring [3]. For the first time, the CtFDO structure reveals a His–Ser active-site pair in the active-site pocket accessible from the exterior via a wide open tunnel. Moreover the active-site pocket is extended by an unusual, mainly hydrophobic, side-cavity.

The GMC family enzymes catalyse the oxidation of primary and secondary alcohols yielding aldehydes or ketones. The measurements of CtFDO catalytic activity with over 1100 compounds did not lead to identification of any strongly reacting substrate. CtFDO appears to be inactive also with common substrates of GMC family enzymes. To get a better insight into the possible substrate moieties and their organization, we performed co-crystallization and crystallographic fragment screening. Five determined structures of complexes with aromatic compounds reveal the potential substrate is of more complex polyaromatic nature.

1. A.-N. Li and D.-C. Li, J. Appl. Microbiol., 106, (2009), 369-380.

2. W. P. Dijkman, G. de Gonzalo, A. Mattevi, M. Fraaije, Appl. Microbiol. Biotechnol., 97, (2013), 5177-5188.

3. L. Sützl, G. Foley, E. M. J. Gillam, M. Bodén, D. Haltrich, Biotechnol Biofuels, 12, (2019), 1-18.

This work is supported by the European Regional Development Fund (CZ.02.1.01/0.0/0.0/15_003/0000447, CZ.02.1.01/0.0/0.0/16_013/0001776, and CZ.1.05/1.1.00/02.0109), institutional support of the Institute of Biotechnology of the Czech Academy of Sciences, v. v. i. (RVO: 86652036), by the Ministry of Education, Youth, and Sports of the Czech Republic (LM2015043, support of Biocev-CMS), and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS19/189/OHK4/3T/14.