Differences in the Activities of Domain‐Swapped Chimeras of Two Homologous GH57 Glucanotransferases Suggest That a Glucan‐Binding DUF Could Influence Donor Substrate Specificity

Eight enzymes of the GH57 family of dis‐proportionating 4‐α‐glucanotransferases have been structurally characterized till date, including five of archaeal hyperthermophilic origin, and three of bacterial origin. Our focus here lies upon three homologous members of this family: (i) PfuAmyGT from Pyrococcus furiosus (PF0272), (ii) TonAmyGT from Thermococcus onnurenius (B6YUX8), and (iii) TliAmyGT (TLGT) from Thermococcus litoralis (O32462). The polypeptide chain of each of these enzymes is approximately 655 residues long, folded into three distinct domains (1, 2, and 3), and assembled into a homodimer. Domain 1 is an incomplete (β/α) 7 barrel containing an aspartate known to function as a catalytic nucleophile in TLGT. Domain 2 (which is helical) and Domain 3 (made up of β sheets) are thought to be domains of unknown function (or DUFs), although some studies indicate that Domain 2 contains residues that influence activity. In PfuAmyGT and TonAmyGT, we have recently identified a catalytically‐important aspartate upon a loop in Domain 2. Further, in PfuAmyGT, we have recently demonstrated the presence of two additional catalytically‐important (glutamate) residues in Domain 1. In this paper, our main focus lies upon Domain 3 which hosts a second binding site (SBS) for a glucan, at its domain‐domain interface with Domain 2. Using strategies involving studies of both (a) domains (or pairs of contiguous domains) extracted from PfuAmyGT, and (b) chimeric three‐domain enzymes recombining analogous domains between PfuAmyGT and TonAmyGT, we demonstrate that Domain 3 determines the choice of the preferred glucan that acts as a donor in the glucan transfer reaction.