Structural Comparison of a Promiscuous and a Highly Specific Sucrose 6F-Phosphate Phosphorylase

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 6^F-phosphate phosphorylases (SPPs) constitute one of the known substrate specificities. Here, we report the characterization of an SPP from <i>Ilumatobacter coccineus</i> with a far stricter specificity than the previously described promiscuous SPP from <i>Thermoanaerobacterium thermosaccharolyticum</i>. 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>I. coccineus</i> were designed. These results contribute to an expanded structural knowledge of enzymes in subfamily GH13_18 and facilitate their rational engineering.