Identification of Thermotoga maritima MSB8 GH57 α-amylase AmyC as a glycogen-branching enzyme with high hydrolytic activity

Abstract

AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary structural comparison suggest that AmyC could actually be a glycogen-branching enzyme (GBE), a key enzyme in the biosynthesis of glycogen. This communication presents phylogenetic and biochemical evidence that AmyC is a GBE with a relatively high hydrolytic (α-amylase) activity (up to 30% of the total activity), creating a branched α-glucan with 8.5% α-1,6-glycosidic bonds. The high hydrolytic activity is explained by the fact that AmyC has a considerably shorter catalytic loop (residues 213–220) not reaching the acceptor side. Secondly, in AmyC, the tryptophan residue (W 246) near the active site has its side chain buried in the protein interior, while the side chain is at the surface in Tk1436 and Tt1467 GBEs. The putative GBEs from three other Thermotogaceae , with very high sequence similarities to AmyC, were found to have the same structural elements as AmyC, suggesting that GH57 GBEs with relatively high hydrolytic activity may be widespread in nature. Electronic supplementary material The online version of this article (10.1007/s00253-019-09938-1) contains supplementary material, which is available to authorized users.

Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes

“…horokoshii GBE, the flexible loop is absent in the T. maritima GBE (Fig. 3 and 5), as was already noted by Zhang and coworkers 8 . It has been reported that AmyC, in spite of the lack of a flexible loop, has a low but reproducible branching activity towards amylose 8 .…”

“…3 and 5), as was already noted by Zhang and coworkers 8 . It has been reported that AmyC, in spite of the lack of a flexible loop, has a low but reproducible branching activity towards amylose 8 . Shortening of the flexible loop in the P. horokoshii GBE from 19 to 9 amino acids resulted in a twofold increase in total activity and a considerable reduction of the branching activity 19 .…”

“…In all the other sequences of this study, three of these four aromatic amino acids are functionally conserved while the F23 is not conserved. Zhang et al 8 reported another three important amino acids near the active site, H11, S462, and D463 (T. thermophilus numbering). These are all conserved at the respective positions in all the 2,497 sequences.…”

“…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 solved 6,10,19,20 . 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-glucans 22 .…”

“…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 chain 1,[5][6][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 acceptors 8 , or perform a cyclization reaction resulting in branched cyclic glucans 9 .…”