Abstract
Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE)
catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked
glucooliogosaccharides. As an atypical family, a limited number of
GH57GBEs have been biochemically characterized so far. This study aimed
at acquiring a better understanding of the GH57GBE family by a
systematic sequence-based bioinformatics analysis of almost 2,500 gene
sequences and determining the branching activity of several native and
mutant GH57GBEs. A correlation was found between a very low or even no
branching activity with the absence of a flexible loop, a tyrosine at
the loop tip, and two β-sheets.
Key words: Glycogen branching enzymes, Glycoside hydrolase
Family 57, Glycogen, Amylose
Introduction
Glycogen branching enzymes (GBE) (EC 2.4.1.18) play a key role in the
biosynthesis of glycogen, a dendrimeric polyglucose carbon and energy
storage molecule present in many prokaryotic microorganisms, fungi,
yeast, and animals1-4. GBE catalyze the formation of
α-1,6 glycosidic linkages in glycogen by transglycosylating a
cleaved-off α-glucan chain from a growing α-1,4 linked
anhydroglucopyranose chain to the same or a different
chain1, 5-7. In addition to the transglycosylating or
branching reaction, GBEs also catalyze the hydrolysis of an α-1-4
glycosidic linkage using water as an acceptors8, or
perform a cyclization reaction resulting in branched cyclic
glucans9.
Based on the primary amino acid sequence and conserved motifs, all known
GBEs are categorized in either the glycoside hydrolase (GH) family 13 or
5710-13. The GBEs from GH13 have a substantially high
activity on the model substrate amylose, a typical linear
α-glucan14-17. GH13 GBEs are involved in the classical
glycogen biosynthetic pathway by the tandem action of
glucose-1-phosphate adenylyltransferase (glgC ) - glycogen
synthase (glgA ) - glycogen branching enzyme
(glgB )2, 18. In contrast to GH13 GBEs, the role
of GH57 GBEs is much less clear. So far, the biochemical properties of
only five GH57 GBEs have been reported, while the crystal structure of
only four of these five GH57 GBEs has been solved6, 10,
19, 20. The activity of these five GH57 GBEs on amylose is relatively
low to almost zero, ranging from a few mU/mg (Thermotoga maritimaSMB8) to a few hundred mU/mg (Thermus thermophilus HB8,Thermococcus kodakarensis KOD1)8, 21. The GH57
GBE of Mycobacterium tuberculosis , having all the key features of
GH57 GBEs, was reported to have no detectable activity on a range of
α-glucans22. Based on the genomic organization, it is
assumed that this GBE branches glucosylglycerate and plays a role in the
biosynthesis of polymethylated polysaccharides22-24.
GH57 GBEs have a triangular three-dimensional shape consisting of three
domains including a catalytic (β/α)7 barrel containing
the two catalytic residues, a glutamate nucleophile and an aspartate
acid/base catalyst6, 10, 19, 20. All GH 57 GBEs have
five conserved sequence regions (CSR), with the nucleophile located in
CSRIII and the acid/base catalyst located in CSRIV25.
Similar to GH13 GBEs, GH57 GBEs employ a double-displacement reaction
mechanism resulting in retention of the α-configuration in the
products6. In the crystal structures of the P.
horikoshii , T. kodakarensis, and T. thermophilus GBE a
flexible loop with a conserved tyrosine at the tip was
identified6, 10, 19. Mutational analysis showed that
the flexible loop and the tyrosine play a key role in the branching
activity; replacing the tyrosine with an alanine in the T.
thermophilus GBE or shortening the loop in P. horokoshii GBE
resulted in a loss of the branching activity6, 19.
The simultaneous presence of a gene encoding a GH13 GBE and one encoding
a GH57 GBE in the genomes of a large number of bacteria makes the
physiological role of GH57 GBEs even more puzzling21.
To gain more insight into the enzymatic activity and possible
physiological role of GH57 GBEs, an in-depth sequence-based
bioinformatics analysis of almost 2,500 GH57 GBE sequences and a basic
biochemical characterization of a number of carefully selected GH57 GBEs
overexpressed in Escherichia coli was conducted. Surprisingly,
the flexible loop covering the active site was absent in the vast
majority of the GH57GBE sequences analyzed in this study. Several of
these loop-deficient GBEs displayed very low to no activity on amylose.
Besides the flexible loop an additional structural element, two adjacent
beta sheets, was identified to play a key role in the branching
activity. It is proposed that GH57 GBEs devoid of the flexible loop and
the two beta sheets are not glycogen branching enzymes and do not play a
role in glycogen biosynthesis but in one or more yet to be identified
metabolic pathways.