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Kuhaudomlarp, Sakonwan, Patron, Nicola J., Henrissat, Bernard, Rejzek, Martin, Saalbach, Gerhard and Field, Robert A. 
ORCID: https://orcid.org/0000-0001-8574-0275
(2018)
Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149.
Journal of Biological Chemistry, 293 (8).
pp. 2865-2876.
ISSN 0021-9258
Abstract
Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain.β-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a β-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro-7066 in E. coli and confirmed it as aβ-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro-7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro-7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial β-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.
| Item Type: | Article |
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| Additional Information: | Funding Information: This work was supported by the BBSRC Institute Strategic Program on Under-standing and Exploiting Metabolism (MET) (Grant BB/J004561/1); the BBSRC, EPSRC, and InnovateUK: IBCatalyst (Grant BB/M02903411); the John Innes Foundation; and the Royal Thai Government Scholarship pro-gram. The authors declare that they have no conflicts of interest with the contents of this article. Author’s Choice—Final version free via Creative Commons CC-BY license. This article contains Tables S1–S3, Figs. S1–S9, and Files S1–S4. The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) MG516599. 1 To whom correspondence should be addressed. E-mail: rob.field@jic.ac.uk. Publisher Copyright: © 2018 by The American Society for Biochemistry and Molecular Biology, Inc. |
| 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: | 04 Sep 2024 08:34 |
| Last Modified: | 25 Sep 2024 18:06 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/96506 |
| DOI: | 10.1074/jbc.RA117.000936 |
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