Crystal Structures of the sugar epimerase for rare sugar production in complexes with deoxy sugars

Transcription

1 The nd International Symposium Space Science of High Quality Protein Crystallization Technology Crystal Structures of the sugar epimerase for rare sugar production in complexes with deoxy sugars ~ Crystal structures of Pseudomonas cichorii D-tagatose -epimerase CS in complexes with deoxy sugars~ Hiromi Yoshida Molecular Structure Research Group Life Science Research Center and Faculty of Medicine Kagawa University, Japan October, 0

2 Outline Introduction What is rare sugar? Bio-production strategy of rare sugars by isomerase/epimerase in Kagawa University Structure of the enzyme for rare sugar production Crystal structures of Pseudomonas cichorii D-tagatose -epimerase (D-TE) Active site structures Substrate recognition and catalytic mechanism Active site structure comparisons in complexes with substrates using crystals grown on the earth and in microgravity Substrate recognition by enzymes including deoxy sugars

3 Rare Sugars Rare sugars are monosaccharides which exist in extremely small amount in nature, but there are a lot of types. D-Galactose D-Fructose D-Xylose D-Mannose D-Ribose D-Allose D-Allulose Xylitol Rare sugar Small amount D-Glucose non-rare sugar L-Arabinose Large amount An image of the ratio of sugars in nature

4 Physiological effect of D-allulose and D-allose Attenuating effect on increase of blood glucose level 血糖値上昇の抑制効果 Reducing effect on visceral fat accumulation 内臓脂肪の蓄積の抑制効果 Antioxidant effect 抗酸化作用 Suppression of cancer cell proliferation 癌細胞の増殖抑制効果 H COH CH OH D-fructose non-rare sugar D-TE 0 kda Homo dimer (0 kda) P. cichorii D-tagatose -epimerase H COH CH OH D-allulose (D-psicose) Rare sugar L-RhI 7 kda Homo tetramer (88 kda) P. stutzeri L-rhamnose isomerase H CH OH D-allose Rare sugar How do the enzymes recognize rare sugars specifically?

5 Overall structure and monomer structure of D-TE Rare Sugars 0 kda Homo dimer (0 kda) (b/a) 8 barrel manganese ion (Mn + ) D-tagatose D-sorbose D-fructose D-allulose Pseudomonas chichorii D-tagatose -epimerase 90 amino acid residues R = 0.7 at.79 Å resolution Yoshida et al. (007). J. Mol. Biol. 7, -

6 Active site structures with bound D-tagatose and D-fructose Asp8 His88 Arg7 Asp8 His88 Arg7 His Phe8 His Phe8 Leu08 Phe7 Trp Trp Leu08 Phe7 Trp Trp Cys.8 Å (PDB: QUM) D-tagatose Loose recognition Cys D-fructose P. cichorii D-TE / D-tagatose P. cichorii D-TE / D-fructose.0 Å (PDB: QUN) Yoshida et al. (007). J. Mol. Biol. 7, -

7 Catalytic reaction mechanism of D-TE Mn + His88 D-fructose Based on the ene-diol mechanism, C-O proton exchange mechanism. H Mn + H H O O C C O H H Intermediate D-allulose D-fructose D-fructose C-O proton exchange mechanism Yoshida et al. (007). J. Mol. Biol. 7, - Yoshida et al., in press, Appl. Microbiol. Biotechnol.

8 Recognition mechanism of deoxy sugars by D-TE D-tagatose D-fructose Rare Sugars D-TE D-TE D-sorbose D-allulose Substrates of D-TE -deoxy -D-tagatose -deoxy -D-tagatose -deoxy -L-tagatose -deoxy -L-tagatose -deoxy -keto-d-galactitol Asp8 His Leu08 Metal Wat His88 Cys Arg7 Phe8 Phe7 D-TE/D-tagatose Strict recognition Trp Trp -deoxy L-allulose -deoxy D-tagatose -deoxy L-tagatose D-tagatose D-sorbose -deoxy keto-d-galactitol

9 Active site structures with bound deoxy sugars -deoxy L-allulose Glu Asp8 His His88 Arg7 Glu8 Trp -deoxy D-tagatose : Metal : Water Ser Leu08 Trp -deoxy L-allulose Ser -deoxy D-tagatose D-TE CS/ -deoxy L-allulose sa omit map at. s.80 Å (PDB: YTT) D-TE CS/ -deoxy D-tagatose sa omit map at s.90 Å (PDB: YTQ) Yoshida et al., in press, Appl. Microbiol. Biotechnol.

10 Active site structures with bound deoxy sugars Asp8 (a) Form A (b) Form B His88 Arg7 His Leu08 Glu8 Cys D-tagatose : Metal : Water Trp Ser Ser D-TE CS/ -deoxy D-tagatose -deoxy D-tagatose D-TE/ D-tagatose -deoxy D-tagatose

11 Crystals grown on the earth or in microgravity Crystallization Capillary counter-diffusion method at 9K, using the following reservoir solution. % (w/v) PEG M Sodium acetate ph. Data collection and structure determination -0 (w/v) deoxy rare sugar solutions were used as cryoprotectant, and structures were determined by molecular replacement. Crystals of D-TE CS grown on the earth Crystals of D-TE CS grown in microgravity

12 Active site structures with bound deoxy sugars Form B Asp8 His88 Arg7 Crystals grown on the earth His Glu8 CysSer Leu08 : Metal : Water -deoxy L-tagatose sa omit map at. s.9 Å (PDB: YTR) Trp PcDTE CS/ -deoxy L-tagatose (molc) CysSer -deoxy L-tagatose

13 Active site structures with bound deoxy sugars His Asp8 CysSer Ser7 : Metal : Water His88 Arg7 Glu8 Trp Trp -deoxy L-tagatose CysSer Ser7 PcDTE CS/ -deoxy L-tagatose (mola) sa omit map at. s.7 Å (PDB: YVL) Form B Crystals grown in microgravity -deoxy L-tagatose Yoshida et al., in press, Appl. Microbiol. Biotechnol.

14 Catalytic reaction mechanism of D-TE

15 Catalytic reaction mechanism of D-TE His88 Mn + H H H O O C C O H Intermediate D-allulose D-fructose C-O proton exchange mechanism Yoshida et al. (007). J. Mol. Biol. 7, - Yoshida et al., in press, Appl. Microbiol. Biotechnol.

16 D-TE Acknowledgements Prof. Shigehiro Kamitori Research assistant Misa Teraoka Molecular Structure Research Group Life Science Research Center and Faculty of Medicine, Kagawa University Prof. Ken Izumori Associate Prof. Goro Takada Associate Prof. Kenji Morimoto Assistant Prof. Akihide Yoshihara Student Takeyori Nishitani International Institute of Rare Sugar Research and Education and Faculty of Agriculture, Kagawa University Prof. Tomohiko Ishii Graduate Student Yasuhiro Tahara Shizuka Kayahara Shunsuke Ohga Department of Advanced Materials Science Faculty of Engineering, Kagawa University Dr. Koji Inaka Dr. Naoki Furubayashi Maruwa Foods and Biosciences Inc. Dr. Hiroaki Tanaka Confocal Science Inc. Dr. Kazunori Ohta Dr. Mitsugu Yamada Japan Aerospace Exploration Agency (JAXA) This study was supported in part by Grant-in-Aid for Young Scientist from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the fund for Characteristic Prior Research and Young Scientists from Kagawa University. This study is contributed by a part of High-Quality Protein Crystal Growth Experiment on KIBO promoted by JAXA (Japan Aerospace Exploration Agency). Russian Space craft Progress or/and Soyuz provided by Russian Federal Space Agency were used for space transportation. A part of space crystallization technology had been developed by European Space Agency and University of Granada.

17 Thank you for your attention!