Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes.
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 beta4-alpha4 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.
Kim
Kwangsoo
K
School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea.
Kim
Hye-Jung
HJ
Oh
Deok-Kun
DK
Cha
Sun-Shin
SS
Rhee
Sangkee
S
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D005632:Fructose / Q000378:metabolism*; D008024:Ligands; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D019998:Racemases and Epimerases / Q000737:chemistry*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D008024:Ligands; D008670:Metals; D050505:Mutant Proteins; C003243:psicose; D005632:Fructose; D019998:Racemases and Epimerases