3.3. The two beta sheets
The second gap in the sequence alignment is in the β5-β6 region, where in the T. thermophilus GBE two prominent beta sheets (β5-β6; 188-205, T. thermophilus GBE numbering), are localized6. Without exception, all 2,296 GBEs (92 %) that miss the flexible loop also do not have the β5 nor β6 sheet (Table 4). In contrast, the majority of the 201 sequences that have a flexible loop also possess the β5-β6 region (159 or 79.1%). Four of the GBEs with a flexible loop and the β5-β6 region were included in this study and showed considerable branching activity ranging from 168 mU/mg to 550 mU/mg. Of those GBEs with a flexible loop but without the β5-β6 region, one, that from Meiothermus sp. PNK-Is4, was overproduced and analysed. It was active towards amylose but as other GBEs without the two beta sheets, it only had a very low branching activity (29.1 mU/mg). Also, the branching over hydrolysis ratio was very similar to those GBEs that missed the β5-β6 region; e.g. K. pacifica and AmyC with a branching over hydrolysis ratio of 3.8 and 5.7 respectively. A similar result was found for the T. hugenholzii GBE, having a long flexible loop of 36 amino acids with a tyrosine and a very short β5-β6 region (Table 3). No activity could be detected for the T. lipolytica GBE, which has a long flexible loop of 28 residues without a tyrosine and no β5-β6 region. Removing the β5-β6 region from theT. kodakarensis GBE resulted in a complete loss of activity (Table 3).
Interestingly, the Mycobacterium tuberculosis H37Rv GH57 GBE also misses the flexible loop as well as the β5-β6 region. Gusthart overexpressed the M. tuberculosis GH57GBE but did not find any activity22. When incubating the M. tuberculosisGH57GBE with amylose, no activity was detected (Gang and van der Maarel, unpublished results). The β5-β6 region thus plays a determining role in the activity of GH57 GBEs, in particular in the branching activity. How exactly this beta sheet region exerts its influence on the branching activity remains unclear.