Applied Microbiology and Biotechnology

, Volume 100, Issue 24, pp 10403–10415 | Cite as

X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates

  • Hiromi Yoshida
  • Akihide Yoshihara
  • Tomohiko Ishii
  • Ken Izumori
  • Shigehiro Kamitori
Biotechnologically relevant enzymes and proteins

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.

Keywords

β/α-Barrel Deoxy sugar Epimerase Rare sugar X-ray structure 

Supplementary material

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Fig. S1(PDF 218 KB)
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Fig. S2(PDF 611 KB)
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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Life Science Research Center and Faculty of MedicineKagawa UniversityKita-gunJapan
  2. 2.Rare Sugar Research Center and Faculty of AgricultureKagawa UniversityKita-gunJapan
  3. 3.Faculty of EngineeringKagawa UniversityTakamatsuJapan

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