Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with β-1,3-endoglucanase activity and founding member of the subfamily GH50_3

Jonathan K Covington; Nicole Torosian; Allison M Cook; Marike Palmer; Scott G Bryan; Nancy O Nou; Ritesh Mewalal; Miranda Harmon-Smith; Ian K Blaby; Jan-Fang Cheng; Matthias Hess; Phillip J Brumm; Nitin K Singh; Kasthuri Venkateswaran; Brian P Hedlund

doi:10.3389/fmicb.2024.1355444

Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with β-1,3-endoglucanase activity and founding member of the subfamily GH50_3

Front Microbiol. 2024 Apr 25:15:1355444. doi: 10.3389/fmicb.2024.1355444. eCollection 2024.

Authors

Jonathan K Covington¹, Nicole Torosian¹, Allison M Cook¹, Marike Palmer^{1

2}, Scott G Bryan¹, Nancy O Nou¹, Ritesh Mewalal³, Miranda Harmon-Smith³, Ian K Blaby³, Jan-Fang Cheng³, Matthias Hess⁴, Phillip J Brumm⁵, Nitin K Singh⁶, Kasthuri Venkateswaran⁶, Brian P Hedlund^{1

7}

Affiliations

¹ School of Life Sciences, University of Nevada, Las Vegas, NV, United States.
² Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
³ US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
⁴ Department of Animal Science, College of Agricultural and Environmental Sciences, University of California, Davis, Davis, CA, United States.
⁵ C5-6 Technologies LLC, Fitchburg, WI, United States.
⁶ Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, United States.
⁷ Nevada Institute of Personalized Medicine, University of Nevada, Las Vegas, NV, United States.

Abstract

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a β-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as β-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high k_cat/K_M of 1.82 × 10⁷ s^-1 M^-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing β-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

Keywords: Armatimonadota; CAZyme; GH50; carboxymethyl curdlan; glycoside hydrolase; hyperthermophile; β-1,3-endoglucanase; β-Glucan.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This material is based upon work supported by the National Aeronautics and Space Administration under Grants No. 80NSSC20M0043, 80NSSC17K0548, 80NSSC21M0157, the US National Science Foundation under grants DBI REU 1757316 and HRD-1712523, and the UNLV Office of Undergraduate Research. The work (proposal: https://doi.org/10.46936/10.25585/60001337) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. The publication fees for this article were supported by the Graduate & Professional Student Association (GPSA).