**Jorick Franceus 1,\*, Nikolas Capra 2, Tom Desmet <sup>1</sup> and Andy-Mark W.H. Thunnissen <sup>2</sup>**


Received: 17 July 2019; Accepted: 8 August 2019; Published: 11 August 2019

**Abstract:** In family GH13 of the carbohydrate-active enzyme database, subfamily 18 contains glycoside phosphorylases that act on α-sugars and glucosides. Because their phosphorolysis reactions are effectively reversible, these enzymes are of interest for the biocatalytic synthesis of various glycosidic compounds. Sucrose 6F-phosphate phosphorylases (SPPs) constitute one of the known substrate specificities. Here, we report the characterization of an SPP from *Ilumatobacter coccineus* with a far stricter specificity than the previously described promiscuous SPP from *Thermoanaerobacterium thermosaccharolyticum*. Crystal structures of both SPPs were determined to provide insight into their similarities and differences. The residues responsible for binding the fructose 6-phosphate group in subsite +1 were found to differ considerably between the two enzymes. Furthermore, several variants that introduce a higher degree of substrate promiscuity in the strict SPP from *I. coccineus* were designed. These results contribute to an expanded structural knowledge of enzymes in subfamily GH13\_18 and facilitate their rational engineering.

**Keywords:** GH13\_18; sucrose phosphorylase; glycoside phosphorylase; *Ilumatobacter coccineus*; *Thermoanaerobacterium thermosaccharolyticum*; crystallography
