**1. Introduction**

Glycoside phosphorylases (GPs) are carbohydrate-active enzymes that catalyze the reversible cleavage of glycosidic bonds using inorganic phosphate [1–3]. In their physiological context, they provide a degradative route for carbohydrates and glycosides that is more energy-efficient when compared to hydrolysis. Their glycosyl phosphate reaction products can enter various pathways, such as glycolysis, without prior activation by a kinase, saving one molecule of ATP [4]. Because of the reversibility of the phosphorolytic reaction, GPs are also attractive enzymes for the synthesis of glycosidic bonds in vitro. One of the most well-studied GPs in that regard is sucrose phosphorylase (SP; EC 2.4.1.7), which catalyzes the phosphorolysis of sucrose into α-d-glucose 1-phosphate (Glc1P) and d-fructose. Thanks to the high energy content of sucrose and the enzyme's remarkable substrate promiscuity, SP is often applied as a catalyst in cost-effective transglucosylation reactions using the cheap and renewable substrate sucrose as a glucosyl donor [5–10]. Moreover, SP has been a target in various engineering studies to further alter its specificity, selectivity, and thermostability [11–15].

Sucrose phosphorylase is found in subfamily 18 of glycoside hydrolase family GH13 (GH13\_18) according to the Carbohydrate-Active Enzyme database (http://www.cazy.org) [16,17]. While searching this subfamily for more thermostable SPs, we previously came across a peculiar putative SP from the thermophile *Thermoanaerobacterium thermosaccharolyticum*. Although the enzyme did show significant activity on sucrose, as was expected, its kinetic parameters revealed that sucrose is in fact not its native substrate. Instead, a clear preference for sucrose 6F-phosphate was observed, making it the first sucrose

6F-phosphate phosphorylase (SPP; EC 2.4.1.329) ever reported [18]. A few key residues responsible for the difference in specificity were identified through homology modeling and mutagenesis. Furthermore, the *T. thermosaccharolyticum* SPP (TtSPP) was shown to accept an even wider range of substrates than the known SPs, offering opportunities for biotechnological applications and further engineering. For instance, a mutant of TtSPP was rationally designed to achieve high activity towards bulky phenolics such as resveratrol, enabling the quantitative production of a resveratrol glucoside [19]. The same mutant could also be applied for the synthesis of glucosylated 3-hydroxy-β-lactams [20].

The discovery of TtSPP hinted at a broader functional diversity in the family left to uncover and triggered the search for other novel GH13\_18 enzymes in recent years. By characterizing proteins from unexplored branches of the phylogenetic tree, phosphorylases from *Meiothermus silvanus*, *Spirochaeta thermophila*, and *Escherichia coli* were found to be dedicated to the substrate 2-*O*-α-d-glucosylglycerate (EC 2.4.1.352) [21]. Further, a comparison of distinctive active site sequence motifs in different branches of the tree led to the discovery of a *Marinobacter adhaerens* enzyme that acts as a configuration-retaining 2-*O*-α-d-glucosylglycerol phosphorylase (EC 2.4.1.359) [22]. In this work, we report the expression and characterization of an SPP from *Ilumatobacter coccineus* YM16–304 (IcSPP) with active site sequence motifs that are different from the motifs found in TtSPP or in the phosphorylases with other specificities in the GH13\_18 subfamily. The enzyme shows high activity on sucrose 6F-phosphate, but unlike TtSPP, it does not catalyze reactions with alternative substrates. Crystal structures of the two SPPs were determined to allow for a comparison of their active site. Automated docking and mutational analyses were carried out to obtain variants of IcSPP with a less stringent specificity.
