d-Psicose, a rare sugar produced by the enzymatic reaction of d-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of d-fructose and d-psicose by epimerizing the C-3 position, with marked efficiency for d-psicose. The crystal structures of DPEase and its complex with the true substrate d-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of d-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the β4-α4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound d-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.
Bibliographical noteFunding Information:
This work was supported by the Brain Korea 21 project, Korea Research Foundation Grant (KRF-2004-005-F00055), and by a grant (CG2114) from Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea.
- Agrobacterium tumefaciens
- crystal structure
- d-psicose 3-epimerase
- metal-dependent catalysis