BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Exploring the sequence diversity in glycoside hydrolase family
13_18 reveals a novel glucosylglycerol phosphorylase
Jorick Franceus
1
&
Lena Decuyper
2
&
Matthias D’hooghe
2
&
Tom Desmet
1
Received: 31 October 2017 /Revised: 1 February 2018 /Accepted: 6 February 2018 /Published online: 22 February 2018
#
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
In the carbohydrate-active enzyme database, GH13_18 is a family of retaining glycoside phosphorylases that act on α-gluco-
sides. 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.
Keywords Glucosylglycerol phosphorylase
.
Glucosylglycerol
.
Glycoside hydrolase family GH13
.
Sucrose phosphorylase
Introduction
Glycoside phosphorylases are carbohydrate-active enzymes
that catalyse the cleavage of glycosidic bonds with the use
of inorganic phosphate, producing a glycosyl phosphate
(Desmet and Soetaert 2011). They are very similar to glyco-
side hydrolases from a functional perspective, differing only
in their use of phosphate instead of water as nucleophile.
However, this difference does have an important practical
consequence (Desmet et al. 2012). The high-energy content
of the formed glycosyl phosphate causes the reaction to be
reversible, which allows phosphorylases to be conveniently
applied as biocatalysts for the synthesis of valuable glycosides
or oligosaccharides in vitro. Unfortunately, the number of dif-
ferent specificities that have been identified to date is rather
limited (Kitaoka 2015). This disadvantage has prompted the
search for novel phosphorylases.
An example of how the functional diversity of these
enzymes has been successfully explored in recent years is
found in subfamily 18 of glycoside hydrolase family 13
(GH13_18) (Stam et al. 2006;Cantareletal.2009). This
subfamily was long thought to contain only sucrose phos-
phorylases (SP, EC 2.4.1.7), which catalyse the reversible
phosphorolysis of sucrose into α-
D
-glucose 1-phosphate
(Glc1P) and
D
-fructose. However, the quest for a thermo-
stable SP revealed a gene from Thermoanaerobacterium
thermosaccharolyticum that actually encodes a sucrose
6′-phosphate phosphorylase (SPP, EC 2.4.1.329) instead
(Verhaeghe et al. 2014). Furthermore, the phylogenetic tree
of GH13_18 was found to comprise two major branches of
which only one contains all sucrose and sucrose 6′-phos-
phate phosphorylases that have been characterised to date.
It was recently discovered that a protein from Meiothermus
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-018-8856-1) contains supplementary
material, which is available to authorized users.
* Tom Desmet
tom.desmet@ugent.be
1
Centre for Synthetic Biology (CSB), Department of Biotechnology,
Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
2
SynBioC Research Group, Department of Sustainable Organic
Chemistry and Technology, Ghent University, Coupure Links 653,
B-9000 Ghent, Belgium
Applied Microbiology and Biotechnology (2018) 102:3183–3191
https://doi.org/10.1007/s00253-018-8856-1
@Phil_Robichaud
@deepthiw
@JoseServera