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

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

doi:10.3389/fmicb.2024.1355444

Title: 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

Journal Article · 25 April 2024 · Frontiers in Microbiology

DOI: https://doi.org/10.3389/fmicb.2024.1355444 · OSTI ID:2407312

Covington, Jonathan K. ^[1]; Torosian, Nicole ^[1]; Cook, Allison M. ^[1]; Palmer, Marike ^[2]; Bryan, Scott G. ^[1]; Nou, Nancy O. ^[1]; Mewalal, Ritesh ^[3]; Harmon-Smith, Miranda ^[3]; Blaby, Ian K. ^[3]; Cheng, Jan-Fang ^[3]; Hess, Matthias ^[4]; Brumm, Phillip J. ^[5]; Singh, Nitin K. ^[6]; Venkateswaran, Kasthuri ^[6]; Hedlund, Brian P. ^[1]

Univ. of Nevada, Las Vegas, NV (United States)
Univ. of Nevada, Las Vegas, NV (United States); Univ. of Manitoba, Winnipeg, MB (Canada)
USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Univ. of California, Davis, CA (United States)
C5-6 Technologies LLC, Fitchburg, WI (United States)
California Institute of Technology (CalTech), Pasadena, CA (United States). Jet Propulsion Lab. (JPL)

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.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); National Science Foundation (NSF)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2407312

Journal Information:: Frontiers in Microbiology, Journal Name: Frontiers in Microbiology Vol. 15; ISSN 1664-302X

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

References (54)

UCSF ChimeraX : Structure visualization for researchers, educators, and developers Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Protein Science, Vol. 30, Issue 1 https://doi.org/10.1002/pro.3943	journal	October 2020
Structure of a glycoside hydrolase family 50 enzyme from a subfamily that is enriched in human gut microbiome bacteroidetes Giles, Kaleigh; Pluvinage, Benjamin; Boraston, Alisdair B. Proteins: Structure, Function, and Bioinformatics, Vol. 85, Issue 1 https://doi.org/10.1002/prot.25189	journal	November 2016
Purification and characterization of a novel β-agarase, AgaA34, from Agarivorans albus YKW-34 Fu, Xiao Ting; Lin, Hong; Kim, Sang Moo Applied Microbiology and Biotechnology, Vol. 78, Issue 2 https://doi.org/10.1007/s00253-007-1303-3	journal	February 2008
Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type β-agarase producing neoagarobiose Kim, Hee Taek; Lee, Saeyoung; Lee, Dongho Applied Microbiology and Biotechnology, Vol. 86, Issue 1 https://doi.org/10.1007/s00253-009-2256-5	journal	October 2009
Characterization of a thermostable endo-1,3(4)-β-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis Meng, Dong-Dong; Wang, Bing; Ma, Xiao-Qing Applied Microbiology and Biotechnology, Vol. 100, Issue 11 https://doi.org/10.1007/s00253-016-7334-x	journal	February 2016
Structure-based design of agarase AgWH50C from Agarivorans gilvus WH0801 to enhance thermostability Zhang, Pujuan; Zhang, Jinru; Zhang, Lujia Applied Microbiology and Biotechnology, Vol. 103, Issue 3 https://doi.org/10.1007/s00253-018-9540-1	journal	December 2018
Microbiology and geochemistry of great boiling and mud hot springs in the United States Great Basin Costa, Kyle C.; Navarro, Jason B.; Shock, Everett L. Extremophiles, Vol. 13, Issue 3 https://doi.org/10.1007/s00792-009-0230-x	journal	February 2009
Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product Lee, Dong-Geun; Jang, Min Kyung; Lee, Ok-Hee Biotechnology Letters, Vol. 30, Issue 5 https://doi.org/10.1007/s10529-008-9634-4	journal	January 2008
Metagenomics of microbial and viral life in terrestrial geothermal environments Strazzulli, Andrea; Fusco, Salvatore; Cobucci-Ponzano, Beatrice Reviews in Environmental Science and Bio/Technology, Vol. 16, Issue 3 https://doi.org/10.1007/s11157-017-9435-0	journal	June 2017
Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801 Liu, Nan; Mao, Xiangzhao; Yang, Meng World Journal of Microbiology and Biotechnology, Vol. 30, Issue 6 https://doi.org/10.1007/s11274-013-1591-y	journal	January 2014
Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium Temuujin, Uyangaa; Chi, Won-Jae; Park, Jae-Sun Journal of Microbiology, Vol. 50, Issue 6 https://doi.org/10.1007/s12275-012-2478-6	journal	December 2012
Exocellular polysaccharides from cyanobacteria and their possible applications De Philippis, R. FEMS Microbiology Reviews, Vol. 22, Issue 3 https://doi.org/10.1016/S0168-6445(98)00012-6	journal	September 1998
Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir Bioresource Technology, Vol. 128 https://doi.org/10.1016/j.biortech.2012.10.145	journal	January 2013
Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis Gao, Hongliang; Xie, Fangrui; Zhang, Wei Carbohydrate Polymers, Vol. 245 https://doi.org/10.1016/j.carbpol.2020.116486	journal	October 2020
Characterization of an endo-1,3-fucanase from marine bacterium Wenyingzhuangia aestuarii: The first member of a novel glycoside hydrolase family GH174 Liu, Guanchen; Shen, Jingjing; Chang, Yaoguang Carbohydrate Polymers, Vol. 306 https://doi.org/10.1016/j.carbpol.2023.120591	journal	April 2023
Extremely thermophilic microorganisms for biomass conversion status and prospects Blumer-Schuette, Sara E.; Kataeva, Irina; Westpheling, Janet Current Opinion in Biotechnology, Vol. 19, Issue 3, p. 210-217 https://doi.org/10.1016/j.copbio.2008.04.007	journal	June 2008
A first glycoside hydrolase family 50 endo-β-1,3-d-glucanase from Pseudomonas aeruginosa Yi, Ping; Yan, Qiaojuan; Jiang, Zhengqiang Enzyme and Microbial Technology, Vol. 108 https://doi.org/10.1016/j.enzmictec.2017.09.002	journal	January 2018
Structure, preparation, modification, and bioactivities of β-glucan and mannan from yeast cell wall: A review Liu, Yana; Wu, Qin; Wu, Xiangyun International Journal of Biological Macromolecules, Vol. 173 https://doi.org/10.1016/j.ijbiomac.2021.01.125	journal	March 2021
Efficient Secretion of the β-Galactosidase Bgal1-3 via both Tat-Dependent and Tat-Independent Pathways in Bacillus subtilis Ren, Guang-hui; Cao, Li-chuang; Kong, Wei Journal of Agricultural and Food Chemistry, Vol. 64, Issue 28 https://doi.org/10.1021/acs.jafc.6b01735	journal	July 2016
Streamlining the Design-to-Build Transition with Build-Optimization Software Tools Oberortner, Ernst; Cheng, Jan-Fang; Hillson, Nathan J. ACS Synthetic Biology, Vol. 6, Issue 3 https://doi.org/10.1021/acssynbio.6b00200	journal	November 2016
Sediment microbial communities in Great Boiling Spring are controlled by temperature and distinct from water communities Cole, Jessica K.; Peacock, Joseph P.; Dodsworth, Jeremy A. The ISME Journal, Vol. 7, Issue 4 https://doi.org/10.1038/ismej.2012.157	journal	December 2012
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
SignalP 6.0 predicts all five types of signal peptides using protein language models Teufel, Felix; Almagro Armenteros, José Juan; Johansen, Alexander Rosenberg Nature Biotechnology, Vol. 40, Issue 7 https://doi.org/10.1038/s41587-021-01156-3	journal	January 2022
Structural insights into β-1,3-glucan cleavage by a glycoside hydrolase family Santos, Camila R.; Costa, Pedro A. C. R.; Vieira, Plínio S. Nature Chemical Biology, Vol. 16, Issue 8 https://doi.org/10.1038/s41589-020-0554-5	journal	May 2020
ColabFold: making protein folding accessible to all Mirdita, Milot; Schütze, Konstantin; Moriwaki, Yoshitaka Nature Methods, Vol. 19, Issue 6 https://doi.org/10.1038/s41592-022-01488-1	journal	May 2022
Intrinsic dynamic behavior of enzyme:substrate complexes govern the catalytic action of β-galactosidases across clan GH-A Kumar, Rajender; Henrissat, Bernard; Coutinho, Pedro M. Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-46589-8	journal	July 2019
Metabolic and enzymatic elucidation of cooperative degradation of red seaweed agarose by two human gut bacteria Yun, Eun Ju; Yu, Sora; Park, Na Jung Scientific Reports, Vol. 11, Issue 1 https://doi.org/10.1038/s41598-021-92872-y	journal	July 2021
Updating the sequence-based classification of glycosyl hydrolases Henrissat, Bernard; Bairoch, Amos Biochemical Journal, Vol. 316, Issue 2 https://doi.org/10.1042/bj3160695	journal	June 1996
Substrate Recognition and Hydrolysis by a Family 50 exo -β-Agarase, Aga50D, from the Marine Bacterium Saccharophagus degradans Pluvinage, Benjamin; Hehemann, Jan-Hendrik; Boraston, Alisdair B. Journal of Biological Chemistry, Vol. 288, Issue 39 https://doi.org/10.1074/jbc.M113.491068	journal	August 2013
A subfamily roadmap of the evolutionarily diverse glycoside hydrolase family 16 (GH16) Viborg, Alexander Holm; Terrapon, Nicolas; Lombard, Vincent Journal of Biological Chemistry, Vol. 294, Issue 44 https://doi.org/10.1074/jbc.RA119.010619	journal	September 2019
MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization Katoh, Kazutaka; Rozewicki, John; Yamada, Kazunori D. Briefings in Bioinformatics, Vol. 20, Issue 4 https://doi.org/10.1093/bib/bbx108	journal	September 2017
Jalview Version 2--a multiple sequence alignment editor and analysis workbench Waterhouse, A. M.; Procter, J. B.; Martin, D. M. A. Bioinformatics, Vol. 25, Issue 9 https://doi.org/10.1093/bioinformatics/btp033	journal	January 2009
IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era Minh, Bui Quang; Schmidt, Heiko A.; Chernomor, Olga Molecular Biology and Evolution, Vol. 37, Issue 5 https://doi.org/10.1093/molbev/msaa015	journal	February 2020
The carbohydrate-active enzyme database: functions and literature Drula, Elodie; Garron, Marie-Line; Dogan, Suzan Nucleic Acids Research, Vol. 50, Issue D1 https://doi.org/10.1093/nar/gkab1045	journal	November 2021
AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models Varadi, Mihaly; Anyango, Stephen; Deshpande, Mandar Nucleic Acids Research, Vol. 50, Issue D1 https://doi.org/10.1093/nar/gkab1061	journal	November 2021
dbCAN2: a meta server for automated carbohydrate-active enzyme annotation Zhang, Han; Yohe, Tanner; Huang, Le Nucleic Acids Research, Vol. 46, Issue W1 https://doi.org/10.1093/nar/gky418	journal	May 2018
MAFFT-DASH: integrated protein sequence and structural alignment Rozewicki, John; Li, Songling; Amada, Karlou Mar Nucleic Acids Research https://doi.org/10.1093/nar/gkz342	journal	May 2019
New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0 Guindon, Stéphane; Dufayard, Jean-François; Lefort, Vincent Systematic Biology, Vol. 59, Issue 3 https://doi.org/10.1093/sysbio/syq010	journal	March 2010
The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains Summers, Emma L.; Moon, Christina D.; Atua, Renee Acta Crystallographica Section F Structural Biology Communications, Vol. 72, Issue 10 https://doi.org/10.1107/S2053230X16014072	journal	September 2016
The structure of PfGH50B, an agarase from the marine bacterium Pseudoalteromonas fuliginea PS47 Pluvinage, Benjamin; Robb, Craig S.; Jeffries, Roderick Acta Crystallographica Section F Structural Biology Communications, Vol. 76, Issue 9 https://doi.org/10.1107/S2053230X20010328	journal	August 2020
Powdered Chitin Agar as a Selective Medium for Enumeration of Actinomycetes in Water and Soil Hsu, S. C.; Lockwood, J. L. Applied Microbiology, Vol. 29, Issue 3 https://doi.org/10.1128/AM.29.3.422-426.1975	journal	March 1975
Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type -Agarase-Producing Neoagarobiose Temuujin, U.; Chi, W. -J.; Chang, Y. -K. Journal of Bacteriology, Vol. 194, Issue 1 https://doi.org/10.1128/JB.05978-11	journal	October 2011
Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization Crosby, James R.; Laemthong, Tunyaboon; Bing, Ryan G. Applied and Environmental Microbiology, Vol. 88, Issue 21 https://doi.org/10.1128/aem.01302-22	journal	November 2022
Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107 Sugano, Y.; Matsumoto, T.; Kodama, H. Applied and Environmental Microbiology, Vol. 59, Issue 11 https://doi.org/10.1128/aem.59.11.3750-3756.1993	journal	November 1993
Genome- and Community-Level Interaction Insights into Carbon Utilization and Element Cycling Functions of Hydrothermarchaeota in Hydrothermal Sediment Zhou, Zhichao; Liu, Yang; Xu, Wei mSystems, Vol. 5, Issue 1 https://doi.org/10.1128/mSystems.00795-19	journal	January 2020
Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens Chen, Zeng-Weng; Lin, Hui-Jie; Huang, Wen-Cheng AMB Express, Vol. 8, Issue 1 https://doi.org/10.1186/s13568-018-0581-8	journal	March 2018
Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle Kato, Shingo; Sakai, Sanae; Hirai, Miho Microbes and Environments, Vol. 33, Issue 1 https://doi.org/10.1264/jsme2.ME17165	journal	January 2018
FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments Price, Morgan N.; Dehal, Paramvir S.; Arkin, Adam P. PLoS ONE, Vol. 5, Issue 3 https://doi.org/10.1371/journal.pone.0009490	journal	March 2010
Pyrosequencing Reveals High-Temperature Cellulolytic Microbial Consortia in Great Boiling Spring after In Situ Lignocellulose Enrichment Peacock, Joseph P.; Cole, Jessica K.; Murugapiran, Senthil K. PLoS ONE, Vol. 8, Issue 3 https://doi.org/10.1371/journal.pone.0059927	journal	March 2013
Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass Chen, Zhiwei; Pereira, Jose H.; Liu, Hanbin PLoS ONE, Vol. 8, Issue 11 https://doi.org/10.1371/journal.pone.0079725	journal	November 2013
Community structure of thermophilic photosynthetic microbial mats and flocs at Sembawang Hot Spring, Singapore George, Christaline; Lim, Chloe Xue Qi; Tong, Yan Frontiers in Microbiology, Vol. 14 https://doi.org/10.3389/fmicb.2023.1189468	journal	June 2023
A Review of Health-Beneficial Properties of Oats Paudel, Devendra; Dhungana, Bandana; Caffe, Melanie Foods, Vol. 10, Issue 11 https://doi.org/10.3390/foods10112591	journal	October 2021
Laminarin, a Major Polysaccharide in Stramenopiles Chen, Jichen; Yang, Jianchao; Du, Hong Marine Drugs, Vol. 19, Issue 10 https://doi.org/10.3390/md19100576	journal	October 2021
Characterization of a Glycoside Hydrolase Family 50 Thermostable β-agarase AgrA from Marine Bacteria Agarivorans sp. AG17 Nikapitiya, Chamilani; Oh, Chul-Hong; Lee, Young-Deuk Fisheries and aquatic sciences, Vol. 13, Issue 1 https://doi.org/10.5657/FAS.2010.13.1.036	journal	March 2010

Similar Records

Characterization of the thermophilic xylanase Fsa02490Xyn from the hyperthermophile Fervidibacter sacchari belonging to glycoside hydrolase family 10

Journal Article · 2025 · FEBS Open Bio · OSTI ID:2997769

Torosian, Nicole; Covington, Jonathan K.; Cook, Allison M.; +8 more

Genome-guided isolation of the hyperthermophilic aerobe Fervidibacter sacchari reveals conserved polysaccharide metabolism in the Armatimonadota

Journal Article · 2024 · Nature Communications · OSTI ID:2482281

Nou, Nancy O.; Covington, Jonathan K.; Lai, Dengxun; +40 more

Author Correction: Genome-guided isolation of the hyperthermophilic aerobe Fervidibacter sacchari reveals conserved polysaccharide metabolism in the Armatimonadota

Journal Article · 2025 · Nature Communications · OSTI ID:2571802

Nou, Nancy O; Covington, Jonathan K; Lai, Dengxun; +40 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Armatimonadota
CAZyme
GH50
carboxymethyl curdlan
glycoside hydrolase
hyperthermophile
β-1
3-endoglucanase
β-Glucan

Title: 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

Citation Formats

References (54)

Similar Records

Related Subjects