The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from α-1,4-linked glucans or malto-oligosaccharides to other α-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (β/α)8 barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 Å and 2.1 Å resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (β/α)8 barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites −2 and −1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the −2 subsite by the side-chain of Lys151 and the stretch of residues 314–317, providing an explanation for the strict transfer specificity of MTase.