Unraveling the subtleties of β-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families

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 polymyxa ATCC 842 in Escherichia coli . We found 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 encoding β-glucosidases and ATP-binding 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 different CAZy families may work sequentially to degrade oligosaccharides.

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

“…oligosaccharides; no assessment of their donor substrate specificity has been reported to date. [7,15] In selecting sugar phosphates to assess with CDP and Pro_ 7066, we considered the synthesis of fragments of human milk oligosaccharides (HMOs), which have been the focus of recent enzymatic syntheses. [16] The synthesis of HMO-like molecules requires the ability to incorporate galactose, which is a prerequisite for later fucosylation or sialylation.…”

“…In terms of single sugar addition, as opposed to oligo/polymerisation reactions with its natural substrate Glc1P, CDP has been shown to be capable of using the anomeric phosphates of xylose (synthesis of a library of β‐(1,4) hetero‐oligosaccharides), galactose (biocatalytic production of novel glycolipids) and glucosamine (microscale reaction; preparative utility not demonstrated) . We recently identified algal and bacterial β‐1,3‐glucan phosphorylases (e.g., Pro_7066) that are capable of producing β‐1,3‐glucan oligosaccharides; no assessment of their donor substrate specificity has been reported to date …”

Retracing Storage Polysaccharide Evolution in Stramenopila

“…Thus, we were able to assign the enzymes suspected of being responsible for polysaccharide branching in Stramenopila to the GH16 subfamily 2. In addition, we checked a high number of putative beta-glucan candidate metabolic enzymes proposed in the literature for beta-glucan polysaccharide storage metabolism ( Michel et al, 2010 ; O’Neill et al, 2015 ; Kuhaudomlarp et al, 2019 ) and tried to correlate them with the presence of the GT48 and GH16 subfamily 2 ( Table 1 and Supplementary Table S1 ). Astonishingly, we did not find a correlation (absence in several Gyrista) with the GH16 subfamily 3 and 4 sequences despite these having been reported to encode laminarinase activities ( Viborg et al, 2019 ).…”

“…For CAZy families and subfamilies, we indicate with a white background if they were linked to one of the metabolisms. Despite a low correlation for GH161, we kept it because of its putative involvement as stated in Kuhaudomlarp et al (2019). Finally, we have indicated on the first column putative activities of each cazyme.…”

“…Intracellular catabolism of storage polysaccharides is performed either through release of glucose monomers or oligomers (by various hydrolytic activities for both alpha- and beta-glucans), or by phosphorolysis that will produce Glucose-1-phosphate (Glc-1-P), which, unlike glucose monomers, retains one of the two high-energy bonds used during glucan synthesis. In alpha-glucan metabolism, this is performed by GT35 ( Wilson et al, 2010 ) phosphorylases, while for beta-glucan degradation, GH94, GH149, and GH161 have all been reported to perform phosphorolysis ( Kitaoka et al, 2012 ; Kuhaudomlarp et al, 2018 , 2019 ) in vitro. Again, their in vivo functional involvement still requires demonstration.…”