title: X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.
abstract: Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.
authors.affiliation: Life Science Research Center and Faculty of Medicine, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Yoshida
authors.affiliation: Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan.
authors.forename: Akihide
authors.initials: A
authors.lastname: Yoshihara
authors.affiliation: Faculty of Engineering, Kagawa University, Hayashi-cho, Takamatsu, Kagawa, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Ishii
authors.affiliation: Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Izumori
authors.affiliation: Life Science Research Center and Faculty of Medicine, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa, Japan. kamitori@med.kagawa-u.ac.jp.
authors.forename: Shigehiro
authors.identifier: http://orcid.org/0000-0002-3950-3372
authors.initials: S
authors.lastname: Kamitori
mesh_terms: D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003837:Deoxy Sugars / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D003837:Deoxy Sugars; D050505:Mutant Proteins; D002238:Carbohydrate Epimerases; C525624:D-tagatose 3-epimerase, Pseudomonas cichorii
keywords: Deoxy sugar; Epimerase; Rare sugar; X-ray structure; β/α-Barrel