Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149

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.

The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase

“…The conserved structural characteristics include (a) the presence of a tryptophan ‐ asparagine ‐ aspartate (WND) motif (W651, N652, and D653) in the catalytic loop, with D653 as the predicted catalytic residue, although the D653 side chain is rotated 100° around CαCβ bond in comparison to that in PsLBP structure; (b) a conserved arginine ‐ aspartate (RD) motif (R470 and D471), which is involved in the recognition of sugar 1‐phosphate, although in the PsLBP structure, the distance between the aspartate residue (D375) and Glc1P is greater than the hydrogen bonding distance; (c) the conserved histidine (H959), which was predicted to be involved in phosphate recognition, although this residue does not form hydrogen bonds with the sulfate molecule in Pro_7066 or PsLBP structures. These structurally conserved amino acids are in agreement with the sequence alignment which predicted the presence of these residues in the enzyme active site . Bicine (BCN) derived from the crystallization precipitant was bound via hydrogen bonding contacts with R470 and E951, possibly mimicking the interaction of the enzyme active site with the hydroxy groups on C3 and C6 of Glc1P donor (Figure F).…”

“…The two copies of the molecule in the ASU form a biologically relevant homodimer with an interfacial area of ∼4230 Å 2 were calculated by jsPISA . The formation of a homodimer in the crystal structure is consistent with the gel filtration analysis, where Pro_7066 was eluted as a dimer . The domains present within each subunit can be defined as follows: an N‐terminal β‐sandwich (residues 1‐303, yellow), a helical linker region (residues 304‐341, lilac), an (α/α) 6 catalytic domain (residues 342‐1045, green) and a C‐terminal jelly roll domain (residues 1046‐1156, red) (Figure B).…”

“…Recently, we discovered a new glycoside hydrolase family 149 (GH149), which contains sequences from microalgae in class Euglenophyceae and notably from gram‐negative bacteria. We have characterized two enzymes from this family, one from the microalga Euglena gracilis , and the other a bacterial enzyme from a metagenomic library, Pro_7066.…”

Preparative and Kinetic Analysis of β‐1,4‐ and β‐1,3‐Glucan Phosphorylases Informs Access to Human Milk Oligosaccharide Fragments and Analogues Thereof

“…Inorganic phosphate, liberated as a result of glycosylation with Gal1P, can participate in the degradative phosphorolysis of β‐linked glucose disaccharide acceptors leading to in situ production of Glc1P (Figure ii). Given the greater kinetic efficiency with which natural donor Glc1P can be used by CDP and Pro_7066 (Table ), a series of extended glucose‐based oligosaccharides, together with glucose, might be formed in these reactions (Figure iii). Some of these oligosaccharides could become capped with galactose, thus leading to a mixture of products (Figure iv).…”

Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM‐1 towards Donor Substrates Glucose/Mannose 1‐Phosphate by Using X‐ray Crystallography and Saturation Transfer Difference NMR Spectroscopy

“…Following on from our efforts to understand GP structure–function relationships and their application in carbohydrate syntheses, herein we investigated the GH94 laminaribiose phosphorylase from Paenibacillus sp. YM‐1 ( Ps LBP), which has previously been reported for its specificity towards LB (β‐ d ‐glucopyranosyl‐(1→3)‐ d ‐glucopyranose; Scheme A) .…”