Biochemical analysis and the preliminary crystallographic characterization of d-tagatose 3-epimerase from Rhodobacter sphaeroides

Microbial Cell Factories

Research

Songtao Li¹  Qianqian Guo¹  Zhangliang Zhu¹  Jian-Wen Wang¹  Panpan Xu¹  Hui-Min Qin²  Zhengliang Qi²  Fuping Lu³ 

[1] College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People’s Republic of China;Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, People’s Republic of China;Tianjin Key Laboratory of Industrial Microbiology, Tianjin, People’s Republic of China;College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People’s Republic of China;Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, People’s Republic of China;Tianjin Key Laboratory of Industrial Microbiology, Tianjin, People’s Republic of China;College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People’s Republic of China;National Engineering Laboratory for Industrial Enzymes, 300457, Tianjin, People’s Republic of China;

关键词: -Tagatose 3-epimerase;    -Fructose;    -Psicose;    Crystallization;    Structural analysis;    TIM-barrel fold;   

DOI  :  10.1186/s12934-017-0808-4

 received in 2017-08-12, accepted in 2017-11-01,  发布年份 2017

来源: Springer

【 摘 要 】

Backgroundd-Tagatose 3-epimerase epimerizes d-fructose to yield d-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A d-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a d-tagatose 3-epimerase that can epimerize d-fructose to yield d-psicose with a high conversion rate.ResultsThe purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris–HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, d-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of d-fructose which indicates RsDTE’s preference of d-fructose more than any other family enzymes.ConclusionsRsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of d-fructose, regulates the substrate recognition. The research on d-tagatose 3-epimerase or d-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

【 授权许可】

CC BY   
© The Author(s) 2017