X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates
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.
Keywordsβ/α-Barrel Deoxy sugar Epimerase Rare sugar X-ray structure
We thank Mr. Y. Tahara, Ms. S. Kayahara, and Mr. S. Ohga for assisting with protein purification; Mr. K. Yube for his technical assistance with DNA sequencing; and Dr. K. Inaka, Dr. N. Furubayashi, Dr. M. Yamada, and Dr. K. Ohta for assisting with crystallizations under microgravity. This research was performed with the approval of the Photon Factory Advisory Committee and National Laboratory for High Energy Physics, Japan. This study is contributed by a part of “High-Quality Protein Crystal Growth Experiment on KIBO” promoted by JAXA (Japan Aerospace Exploration Agency). Russian spacecraft “Progress” and/or “Soyuz” provided by the Russian Federal Space Agency were used for space transportation. A part of space crystallization technology had been developed by the European Space Agency and University of Granada.
Compliance with ethical standards
This study was funded in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (25440028, 23770122).
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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