3.2 The flexible loop and tyrosine
From the sequence alignment, two major gaps became visible. The first was a sequence of 19 amino acids from position 229 to 247 (T. thermophilus GBE numbering) in between CSR III and IV. In the crystal structures of T. thermophilus 6, T. kodakarensis 10, and P. horokoshii 19 GBE, these amino acids make up a flexible loop covering the catalytic cleft (Fig. 3). In 201 other amino acid sequences, a flexible loop of varying length is present (Fig. 4). The average loop size is 26 amino acids with the shortest being 13 amino acids, while the longest is 50 amino acids. At the tip of the flexible loop of T. thermophilus , T. kodakarensis , and P. horokoshii GBE, a tyrosine is present6, 10, 19. TheT. thermophilus tyrosine was shown to play a prominent role in the branching activity. Replacing this tyrosine with an alanine resulted in a loss of branching activity6. A minority of GBEs with a flexible loop do not have a tyrosine at the tip (35; 17.2%). Instead an alanine, serine, or threonine are found. TheThermoanaerobaculum aquaticum GBE, which has a medium sized loop of 22 residues with an alanine at the tip, has a relatively high activity towards amylose, dominated by the branching activity of 168 mU/mg and a ratio of branching over hydrolytic activity of 38.1 (Table 3). On the contrary, the Calidithermus timidus GBE with a flexible loop of 23 amino acids and a tyrosine at the tip, has a branching activity of 356.9 mU/mg, being twice that T. aquaticumGBE. This result indicates that the tyrosine at the tip of the flexible loop does not play a determining role in the branching activity. This was confirmed by a tyrosine to alanine mutation in the T. kodakarensis GBE, which has a flexible loop of 19 amino acids, and the same size and configuration as that of the T. thermophilus GBE (Table 3; Fig. 3). Whereas the wild type T. kodakarensis GBE has a dominant branching activity of 480 mU/mg and a branching over hydrolysis ratio of 41, the Y/A mutant retains the dominant branching activity (426 mU/mg). The hydrolytic activity of the Y/A mutant doubled from 12.6 mU/mg to 26.3 mU/mg, resulting in a branching over hydrolysis ratio of 16.2. These results suggest that not only the tyrosine at the tip of the loop but also the size and configuration of the flexible loop play a role in the branching activity.
In contrast to the T. thermophilus , T. kodakarensis andP. horokoshii GBE, the flexible loop is absent in the T. maritima GBE (Fig. 3 and 5), as was already noted by Zhang and coworkers8. It has been reported that AmyC, in spite of the lack of a flexible loop, has a low but reproducible branching activity towards amylose8. 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 activity19. The flexible loop is not only absent in AmyC but also in 2,296 of the 2,497 sequences (92%) used in this study (Table 4). A comparable low branching activity was found for the GBE of P. mexicana and Kosmotoga pacifica , of 14.1 mU/mg and 9.1 mU/mg respectively (Table 3). Although having a low activity, both enzymes introduced 6% branches in the final product when incubated with amylose V (data not shown), confirming that both enzymes clearly have branching activity. Introduction of the full or partial flexible loop of T. kodakarensis GBE, including the tyrosine, in the T. maritima GBE resulted in a two-fold increase of the branching activity and a three-fold increase of the hydrolytic activity (Tables 3), being substantially lower than the activity of the wild typeT. kodakarensis GBE. The flexible loop appears not to be the only structural element that determines the overall activity of the GH57 GBEs.
The length of about 19 residues and configuration of the T. thermophilus and T. kodakarensis GBE flexible loop seems to be optimal with respect to the branching activity. Hundred and three of the 201 sequences (51%) have a flexible loop of 17 to 22 amino acids (Fig. 4). A smaller, but still significant number of proteins (81; 40%) have a flexible loop of 24 or more amino acids while 17 proteins (9%) have a flexible loop of 13 to 15 amino acids. The models of the T. hugenholzii and T. lipolytica GBE, with 36 and 28 amino acids loop resp., show that only the first part of the flexible loop folds into the active site cleft while the rest folds next to or behind the part that covers the cleft (Fig. 5). The T. hugenholzii flexible loop contains a tyrosine at position 245 which is turned away from the cleft being far away from the two active site residues (E191 and D382). This GBE has a very low branching activity of only 13.9 mU/mg, while theT. lypolytica GBE did not show any activity (Table 3). Thus, for a GH57 GBE to act as a “true” glycogen branching enzyme, a flexible loop of 17-22 amino acids covering the active cleft and, when present, a tyrosine positioned deep into the active site close to the catalytic E and D is required. The absence of the flexible loop or a loop smaller or larger than 17-22 amino acids results in a significant reduction to complete loss of activity towards amylose. What the in-vivosubstrate for these loop-deficient and long-looped GH57 GBEs is, remains to be established. It could be that these GBEs do show activity towards a growing α-glucan chain, the in-vivo substrate of most GBEs1.