A glucanotransferase that uses two sub-sites and four catalytic aspartates/glutamates to disproportionate oligosaccharides ranging in length from maltotriose to starch

Abstract

AbstractPF0272 (PfuAmyGT) fromPyrococcus furiosusis a 656 residues-long, homodimeric, three-domain GH57 glycoside hydrolase [homologous to TLGT fromThermococcus litoralis(PDB ID: 1K1X)]. It is proposed to be either an α-amylase (EC 3.2.1.1), or a 4-α-glucanotransferase (EC 2.4.1.25). We demonstrate that PfuAmyGT is an exo-amylase-cum-glucanotransferase capable of transferring glucose, and dis-proportionating oligosaccharides, by excising glucose from malto-oligosaccharides (ranging in length from maltotriose to amylose/starch), and transferring it to malto-oligosaccharides (ranging in length from glucose to maltoheptaose and, possibly, even longer lengths). Convention holds that glucanotransferases transfer sugars through the serial and alternating binding of donors and acceptors to the same site, with covalent retention of excised sugars between such bindings. We present evidence of multiple behaviors in PfuAmyGT that challenge this view: (i) Production of free glucose, indicating scope for release of excised glucose; (ii) Higher activity with longer donors, indicating processivity; (iii) Accelerated activity with shorter acceptors, indicating a dependence upon rapid acceptor turnover; and (iv) Evidence of four catalytic glutamates/aspartates (E131, D222, E224, D362), indicating possible separation of excision and ligation functions. These behaviours collectively indicate the binding of donors and acceptors to separate sub-sites that have different substrate ‘length’ preferences, supporting our own previous proposal regarding separate tunnel (internal) and groove (surface) binding sites. Although PfuAmyGT’s mechanism of function remains to be fully elucidated, this paper definitively demonstrate ‘coupling’ of exo-amylase and glucanotransferase functions involving separate sub-sites for donor and acceptor binding.