The Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell

Abstract

Sucrose-phosphatase (SPP) catalyzes the final step in the pathway of sucrose biosynthesis in both plants and cyanobacteria, and the SPPs from these two groups of organisms are closely related. We have crystallized the enzyme from the cyanobacterium Synechocystis sp PCC 6803 and determined its crystal structure alone and in complex with various ligands. The protein consists of a core domain containing the catalytic site and a smaller cap domain that contains a glucose binding site. Two flexible hinge loops link the two domains, forming a structure that resembles a pair of sugar tongs. The glucose binding site plays a major role in determining the enzyme's remarkable substrate specificity and is also important for its inhibition by sucrose and glucose. It is proposed that the catalytic reaction is initiated by nucleophilic attack on the substrate by Asp9 and involves formation of a covalent phospho-Asp9-enzyme intermediate. From modeling based on the SPP structure, we predict that the noncatalytic SPP-like domain of the Synechocystis sucrose-phosphate synthase could bind sucrose-6 F -phosphate and propose that this domain might be involved in metabolite channeling between the last two enzymes in the pathway of sucrose synthesis.

Gene families and evolution of trehalose metabolism in plants

“…The phosphatase domain of all these proteins includes three amino acid motifs that are characteristic of the L-2-haloacid dehalogenase (HAD) superfamily of enzymes: motif I -DX(D/T/Y)X(T/V)(L/V/I), motif II -a Ser or Thr, generally in a hydrophobic context, and motif III -KX 18-30 (G/S)(D/S)X 3 (D/N). Motif I is the most highly conserved, and the invariant first Asp is the functional nucleophile that forms a phospho-acyl intermediate during catalysis (Fieulaine et al 2005). The HAD superfamily embraces a wide range of phosphatases and hydrolases, including the analogous enzyme of sucrose synthesis, sucrosephosphatase (Lunn et al 2000), and crystal structures show that the three conserved motifs form the active site of HAD superfamily enzymes (Fieulaine et al 2005;Burroughs et al 2006).…”

“…Motif I is the most highly conserved, and the invariant first Asp is the functional nucleophile that forms a phospho-acyl intermediate during catalysis (Fieulaine et al 2005). The HAD superfamily embraces a wide range of phosphatases and hydrolases, including the analogous enzyme of sucrose synthesis, sucrosephosphatase (Lunn et al 2000), and crystal structures show that the three conserved motifs form the active site of HAD superfamily enzymes (Fieulaine et al 2005;Burroughs et al 2006). The presence of these motifs in the class II isoforms of TPS prompted speculation that they might have TPP activity (Leyman et al 2001), but no such activity has been found.…”

A metabolic pathway leading to mannosylfructose biosynthesis in Agrobacterium tumefaciens uncovers a family of mannosyltransferases

“…PCC 6803 SPP complexed with Suc-6P, shows that the hexose 2-hydroxyl group of the Suc-6P molecule, the only feature that discriminates Suc-6P from MF-6P (Fig. 1), is not involved in the interactions with the disaccharide binding site (16).…”

Trehalose Metabolites in Arabidopsis—elusive, active and central

“…Sucrose phosphate synthases similarly have a non functional SPP domain which was proposed to serve the purpose of substrate channeling (Fieulaine et al, 2005), but in the case of TPS it could also serve to sequester T6P. The AtTPS1 enzyme is special because of an N-terminal extension, which is not found in other Arabidopsis TPSs and was shown to mediate regulation of TPS activity; it is also found in the SlTPS1 from Selaginella lepidophylla, a desert resurrection plant that accumulates large amounts of trehalose (Van Dijck et al, 2002).…”