Exploring the sequence diversity in glycoside hydrolase family 13_18 reveals a novel glucosylglycerol phosphorylase

Abstract

In the carbohydrate-active enzyme database, GH13_18 is a family of retaining glycoside phosphorylases that act on α-glucosides. In this work, we explored the functional diversity of this family by comparing distinctive sequence motifs in different branches of its phylogenetic tree. A glycoside phosphorylase from Marinobacter adhaerens HP15 that was predicted to have a novel function was expressed and characterised. The enzyme was found to catalyse the reversible phosphorolysis of 2-O-α-D-glucosylglycerol with retention of the anomeric configuration, a specificity that has never been described before. Homology modelling, docking and mutagenesis were performed to pinpoint particular acceptor site residues (Tyr194, Ala333, Gln336) involved in the binding of glycerol. The new enzyme specificity provides additional insights into bacterial metabolic routes, being the first report of a phosphorolytic route for glucosylglycerol in a glucosylglycerol-producing organism. Furthermore, glucosylglycerol phosphorylase might be an attractive biocatalyst for the production of the osmolyte glucosylglycerol, which is currently produced on industrial scale by exploiting a side activity of the closely related sucrose phosphorylase. Family GH13_18 has clearly proven to be more diverse than was initially assumed, and the analysis of specificity-determining sequence motifs has shown to be a straightforward and fruitful tool for enzyme discovery.

Sucrose 6F-phosphate phosphorylase: a novel insight in the human gut microbiome

“…Ms GGaPs, St GGaPs and Ec GGaPs, respectively) catalyse the reversible phosphorolysis of glucosylglycerate into G1P and d -glycerate. Very recently, an enzyme from Marinobacter adhaerens HP15 able to catalyse the reversible phosphorolysis of 2- O -α- d -glucosylglycerol has been characterized and named as glucosylglycerol phosphorylase ( Ma GGoP [27]).…”

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

“…Another novel phosphorylase specificity was discovered by searching GH13_18 for sequences that do not contain the characteristic motif of SP, SPP and GGaP in loop A. Sequences with a peculiar VGAIYQ motif were found in a clade that is interlocked between those of SPs and promiscuous SPPs (Figure 2, Figure S1). Expression and characterisation of an enzyme from Marinobacter adhaerens from this clade revealed that they are strict 2-O-glucosylglycerol phosphorylases (GGoP; EC 2.4.1.359) [18]. Glucosylglycerol is a compatible solute, just like glucosylglycerate, and GGoP is the only known enzyme that can catalyse its phosphorolysis in glucosylglycerol-producing organisms.…”

“…Glu264 and Gln378 were predicted to be binding partners too, but their influence has yet to be verified in vitro. Finally, in the GGaP from M. silvanus and the GGoP from M. adhaerens, a few specificity-determining residues have been uncovered by molecular docking in a homology model followed by mutagenesis [15,18]. Substitution of Asn275 and Glu383 severely decreases the activity of GGaP.…”

A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides

“…Similarly, glucosylglycerol phosphorylase-like sequences can be distinguished from others by the conserved VGA motif in one of the acceptor site loops (loop A; motif at positions 331-333 in the Marinobacter adhaerens enzyme). The alanine residue is predicted to participate in hydrophobic interaction with the C 1' carbon of glucosylglycerol [10]. In the neighbouring clades of sucrose and sucrose 6 F -phosporylases, that alanine is always replaced by aspartate.…”

“…No new specificities from evolutionarily closely related sequences were found until 2014, when Verhaeghe et al [8] described a phylogenetic neighbour that was specialised in the phosphorolysis of sucrose 6 F -phosphate (EC 2.4.1.329), albeit with a very broad substrate specificity. Rational searches for other new functions led to the recent discovery of glucosylglycerate phosphorylases (EC 2.4.1.352) [9], glucosylglycerol phosphorylases (EC 2.4.1.359) [10] and sucrose 6 F -phosphorylases with a strict substrate specificity [11,12].…”