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.