Differences in the activities of domain-swapped chimeras of two homologous GH57 glucanotransferases suggest that a glucan-binding DUF could influence donor substrate specificity

Abstract

Five enzymes of the archaeal hyperthermophilic family of disproportionating GH57 4--glucanotransferases have been studied till date. 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 a beta/alpha barrel containing an aspartate known to function as a catalytic nucleophile in TLGT. Domain 2 (which is helical) and domain 3 (made up of beta sheets) are thought to be domains of unknown function (or DUFs). In PfuAmyGT and TonAmyGT, we have recently identified a catalytically-important aspartate upon a loop in domain 2. In PfuAmyGT, we demonstrate the presence of two additional catalytically-important (glutamate) residues in domain 1, in a companion paper. In this paper, our 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.