Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity analysis

Abstract

The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 2.4.1.49) produces cellodextrin oligomers from short β-1→4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.

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 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). These domains are similar to those observed in PsLBP and other GH94 enzymes (Figure C,D) . Cellodextrin phosphorylase from Ruminiclostridium thermocellum (RtCDP) and β‐(1 → 2)‐oligoglucan phosphorylase from Lachnoclostridium phytofermentans (LpSOGP), both belonging to GH94 family, have additional, but dissimilar, N‐terminal domains (Figure C, purple in RtCDP), but neither is present in Pro_7066.…”

“…Enzymatic synthesis is an attractive approach to such materials due to its regiospecificity and stereospecificity. Emulating what has been achieved with α‐1,4‐glucans and β‐1,4‐glucans, glycoside phosphorylases (GPs) with specificity for β‐(1 → 3)‐glycosidic bonds are potential candidate biocatalysts for such syntheses. GPs acting on β‐ d ‐glucopyranosyl‐(1 → 3)‐ d ‐glucopyranose (laminaribiose) have previously been described in bacteria Paenibacillus sp.…”

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

“…This prompted us to challenge the catalytic capabilities of two wild-type glycoside phosphorylases, [1d] which have yet to find widespread use in the synthesis of smaller glycans, [5] as both enzymes are innate polymerases. Specifically, we investigated the action of b-1,4-glucan linkagedependent cellodextrin phosphorylase (CDP, GH94) [6] and b-1,3-glucan linkage-dependent laminaridextrin phosphorylase (Pro_7066, GH149; [7] natural reactions shown in Figure 1) in reactions with a range of both natural and unnatural sugar-1phosphate donors and glucan acceptor substrates.…”

“…[11,12] 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 b-(1,4) hetero-oligosaccharides), [13] galactose (biocatalytic production of novel glycolipids) [14] and glucosamine (microscale reaction; preparative utility not demonstrated). [6] We recently identified algal and bacterial b-1,3-glucan phosphorylases (e.g., Pro_7066) that are capable of producing b-1,3-glucan…”

“…In order to rationalise the difference in the tolerance towards C2 modification of sugar 1-phosphates by CDP and Pro_ 7066, we compared the reported X-ray crystal structures of the two proteins (PDB IDs: 5NZ8 and 6HQ6, respectively), [6,20] although structures of complexes with donor substrate are not yet available. In the structure of CDP in complex with cellotetraose and phosphate, the hydroxy group at C2 of the nonreducing terminal Glc residue occupying the sugar 1-phosphate binding site (À1 subsite) makes two hydrogen bond contacts with R496 (Figure 2 A, red).…”