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.
Highlights
Glycoside hydrolases (GHs; EC 3.2.1.x) catalyse the hydrolysis of O-glycosidic bonds in carbohydrates such as starch.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Slovak Academy of Sciences, SK-84551 Bratislava, Slovakia 4 Department of Biology, Faculty of Natural Sciences, University of SS Cyril and Methodius, SK-91701 Trnava, Slovakia 5 Biomolecular X-ray Crystallography, Groningen BiomolecularSciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, NetherlandsThey are ubiquitously present in all kingdoms of life
The alignment was done for AmyC from T. maritima and the three characterized glycogen-branching enzyme (GBE) from T. kodakaraensis, T. thermophilus and P. horikoshi, for which their threedimensional structures have been determined—using the program Clustal-Omega with default parameters (Sievers et al 2011)
The eventual α-glucan branching activity was first ascribed to AmyC based on a detailed in silico sequence analysis (Blesak and Janecek 2012) mainly due to presence of a cysteine residue (Cys186) in the conserved sequence regions (CSRs)-3 (Fig. 2), which was suggested to be a clear branching enzyme sequence feature
Summary
Glycoside hydrolases (GHs; EC 3.2.1.x) catalyse the hydrolysis of O-glycosidic bonds in carbohydrates such as starch. The authors proposed that the differences in tertiary and quaternary structure relate to the fact that AmyC only showed hydrolytic activity on starch-like substrates. This hypothesis was further supported by the observation that TT1467 was characterized as a GBE (PDB entry 3P0B (Palomo et al 2011)) and features the same structural elements as TK1436, but differs from AmyC regarding those. A detailed bioinformatic analysis of GH57 enzymes (Blesak and Janecek 2012) clearly showed that AmyC contains the sequence fingerprint of GBE’s; it remained intriguing why the biochemical characterization of AmyC (Ballschmiter et al 2006) only revealed hydrolytic and not transglycosylation (branching) activity. Three putative GH57 GBEs are identified based on structural homology to AmyC, suggesting that GH57 GBEs with relatively high hydrolytic activity are more widespread in mesophilic and thermophilic microorganisms
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