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 a β-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.
Highlights
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. -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 E. gracilis phosphorylase 1 (EgP1), by proteomic analysis of an enriched cellular protein lysate
New -1,3-glucan phosphorylase family lases and their applications in enzymatic synthesis [19, 20], we focused on the established capability of the organism for -1,3D-glucan biochemistry, in particular its known -1,3-D-glucan phosphorylase activities (EC 2.4.1.31) (Fig. 1)
Summary
Identification of Euglena phosphorylase candidates through proteomic analysis It has previously been hypothesized that Euglena -1,3-glucan phosphorylase sequences may belong to GH94 family based on an activity somewhat similar to the GH94 bacterial laminaribiose phosphorylases [26, 27] To test this hypothesis, we used 20 characterized GH94 amino acid sequences as queries (Table S1) to interrogate the translated Euglena transcriptome [14] using tBLASTn (threshold ϭ 0.0001). To investigate whether EgP1 activity is specific to -1,3linked glucan, phosphorolysis assays (Fig. 1) were performed against a variety of glucose disaccharides with various glycosidic linkages and anomeric configurations Analysis of these reactions by TLC and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD) showed that EgP1 was active exclusively on the Glc-1,3-Glc oligosaccharides, confirming its function as a -1,3-D-glucan phosphorylase (Fig. 3, C and D). The Bacteroidetes species from which GH149 were found were predominantly isolated from marine environments; 13 have been described to be associated with algae and phytoplankton
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