Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose.
Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 A, respectively. A subunit of P. cichoriid-TE adopts a (beta/alpha)(8) barrel structure, and a metal ion (Mn(2+)) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the beta-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn(2+), and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.
Yoshida
Hiromi
H
Life Science Research Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Yamada
Mitsugu
M
Nishitani
Takeyori
T
Takada
Goro
G
Izumori
Ken
K
Kamitori
Shigehiro
S
D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D005632:Fructose / Q000737:chemistry*; D006601:Hexoses / Q000737:chemistry*; D006860:Hydrogen Bonding; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D006601:Hexoses; D005632:Fructose; D002238:Carbohydrate Epimerases; C525624:D-tagatose 3-epimerase, Pseudomonas cichorii; C030192:tagatose