New groups of protein homologues in the α-amylase family GH57 closely related to α-glucan branching enzymes and 4-α-glucanotransferases.

The glycoside hydrolase family GH57 is known as the second α-amylase family. Its main characteristics are as follows: (i) employing the retaining reaction mechanism; (ii) adopting the (ß/α)7-barrel (the incomplete TIM-barrel) with succeeding bundle of α-helices as the catalytic domain; (iii) sharing the five conserved sequence regions (CSRs) exhibiting the sequence fingerprints of the individual enzyme specificities; and (iv) using the catalytic machinery consisting of glutamic acid (the catalytic nucleophile) and aspartic acid (the proton donor) positioned at strands ß4 (CSR-3) and ß7 (CSR-4) of the (ß/α)7-barrel domain, respectively. Several years ago, a group of hypothetical proteins closely related to the specificity of α-amylase was revealed, the so-called α-amylase-like homologues, the members of which lack either one or even both catalytic residues. The novelty of the present study lies in delivering two additional groups of the "like" proteins that are homologues of α-glucan-branching enzyme (GBE) and 4-α-glucanotransferase (4AGT) specificities. Based on a recently published in silico analysis of more than 1600 family GH57 sequences, 13 GBE-like and 18 4AGT-like proteins from unique sources were collected and analyzed in a detail with respect to their taxonomical origin, sequence and structural features as well as evolutionary relationships. This in silico study could accelerate the efforts leading to experimental revealing the real function of the enzymes-like proteins in the α-amylase family GH57.

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

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.

In silico analysis of the α-amylase family GH57: eventual subfamilies reflecting enzyme specificities.

Glycoside hydrolases (GHs) have been classified in the CAZy database into 153 GH families. Currently, there might be four α-amylase families: the main family GH13, the family GH57 with related GH119 and, eventually, also GH126. The family GH57 was established in 1996 as the second and smaller α-amylase family. In addition to α-amylase, it contains 4-α-glucanotransferase, α-glucan branching enzyme, amylopullulanase, dual-specificity amylopullulanase-cyclomaltodextrinase, non-specified amylase, maltogenic amylase and α-galactosidase. The family GH57 enzymes employ the retaining reaction mechanism, share five typical conserved sequence regions and possess catalytic (ß/α)7-barrel succeeded by a four-helix bundle with the catalytic machinery consisting of catalytic nucleophile and proton donor (glutamic acid and aspartic acid at strands ß4 and ß7, respectively). The present bioinformatics study delivers a detailed sequence comparison of 1602 family GH57 sequences with the aim to highlight the uniqueness of each enzyme's specificity and all eventual protein groups. This was achieved by creating the evolutionary tree focused on both the enzyme specificities and taxonomical origin. The substantial increase of numbers of sequences from recent comparisons done more than 5 years ago has allowed to refine the details of the sequence logos for the individual enzyme specificities. The study identifies a new evolutionary distinct group of α-galactosidase-related enzymes with until-now-undefined enzyme specificity but positioned on the evolutionary tree on a branch adjacent to α-galactosidases. The specificity of α-galactosidase is, moreover, the only one of the entire family GH57 for which there is no structural support for the proposal of the proton donor based on sequence analysis. The analysis also suggests a few so-called "like" protein groups related to some family GH57 enzyme specificities but lacking one or both catalytic residues.