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Kuhaudomlarp, Sakonwan, Pergolizzi, Giulia, Patron, Nicola J., Henrissat, Bernard and Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275
(2019)
Unraveling the subtleties of β-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families.
Journal of Biological Chemistry, 294 (16).
pp. 6483-6493.
ISSN 0021-9258
Abstract
Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural β-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on β-(1→3)- glucooligosaccharides is an attractive area of research. GP activities acting on β-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized β-(1→3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on β-(1→3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxaATCC 842 in Escherichia coli.Wefound that PapP acts on β-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP)> 2. This activity was distinct from that of characterized GH149 β-(1→3)-glucan phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encodingβ-glucosidases and ATPbinding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from differentCAZyfamilies may work sequentially to degrade oligosaccharides.
| Item Type: | Article |
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| Additional Information: | Funding Information: This work was supported by UK BBSRC Institute Strategic Program Molecules from Nature (MfN) Grant BB/PO12523/1, Open Plant Synthetic Biology Centre Grant BB/LO1413/1, the John Innes Foundation, and the Royal Thai Government Scholarship program. The authors declare that they have no conflicts of interest with the contents of this article. Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. This article contains Tables S1–S3, Figs. S1–S5, and Files S1 and S2. 1Present address: Université Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France. 2 To whom correspondence should be addressed. E-mail: rob.field@jic.ac.uk. Publisher Copyright: © 2019 Kuhaudomlarp et al. |
| Uncontrolled Keywords: | biochemistry,molecular biology,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1303 |
| Faculty \ School: | Faculty of Science > School of Chemistry, Pharmacy and Pharmacology |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 02 Sep 2024 15:31 |
| Last Modified: | 25 Sep 2024 18:05 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96451 |
| DOI: | 10.1074/jbc.RA119.007712 |
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