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

Abstract

Glycoside phosphorylases (GPs) with specificity for β‐(1 → 3)‐gluco‐oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X‐ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β‐(1 → 3)‐gluco‐oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.

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

“…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), 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 (−1 subsite) makes two hydrogen bond contacts with R496 (Figure A, red).…”

Insights to improve the activity of glycosyl phosphorylases from Ruminococcus albus 8 with cello-oligosaccharides

“…Cellobiose and cellodextrin phosphorylases belong to family GH94 together with other glycosyl phosphorylases like laminaribiose phosphorylases (LBPs), chitobiose phosphorylases (ChBPs), and cellobionic acid phosphorylases (CBAPs). They are commonly homodimers, and each subunit is composed of three distinct domains: an N-terminal β-sandwich domain that forms most of the dimer interface, a helical linker, and an (α/α) 6 -barrel catalytic domain (Hidaka et al, 2004(Hidaka et al, , 2006Bianchetti et al, 2011;Nakajima H et al, 2017;Kuhaudomlarp et al, 2019). Interestingly, the length and conformation of catalytic, adjacent, and opposite loops were identified as the determining features that restrict the size of the acceptor binding site differentiating between CBPs, CDPs, and ChBPs (Hidaka et al, 2004;De Groeve et al, 2010;Bianchetti et al, 2011;O'Neill et al, 2017;Gabrielli et al, 2021).…”

Recent advances in enzymatic synthesis of β-glucan and cellulose

“…In order to gain understanding of β-1,3-D-glucan phosphorylases at the molecular level and elucidate the basis for oligosaccharides chain length specificity of GH149 enzymes, the X-ray crystallographic structures of Pro_7066 in the absence of substrate (PDB code 6HQ6 ) and in complex with laminarihexaose (PDB code 6HQ8 ) were solved. Although the overall domain organisation is similar to GH94, Pro_7066 enzyme contains two additional distinct domains flanking its catalytic region and a surface oligosaccharide binding site where laminarihexaose was accommodated, which is distant from the catalytic site and may be involved in the recognition of longer substrates [ 129 ]. The crystallographic structure of the GH94 laminaribiose phosphorylase from Paenibacillus sp YM-1 ( Ps LBP, PDB code 6GH2 ) demonstrated its specificity for disaccharides.…”