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.