Crystal Structural Analysis of DehDIV-<i>R</i> by X-ray Crystallography

doi:10.13523/j.cb.20180803

China Biotechnology

2018, Vol. 38

Issue (8): 19-25 DOI: 10.13523/j.cb.20180803

Crystal Structural Analysis of DehDIV-R by X-ray Crystallography

Chao-di TONG,Jian-ping WU,Li-rong YANG,Gang XU(

)

Zhejiang University,Department of Chemical and Biological Engineering,Hangzhou 310027,China

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Abstract

R-2-haloacid dehalogenase can selectively hydrolyze R-2-haloacid and have important applications in the synthesis of chiral compounds.The analysis of the crystal structure provides a direct structural guide to improve the selectivity and activity of the enzyme,which is the frontier in the field of enzymatic structure research.The crystal structure of R-2-chlorpropionic acid dehalogenase (DehDIV-R) from Pseudomonas ZJU26 was studied.DehDIV-R was expressed in Escherichia coli BL21(DE3) using ppSUMO as vectors,and purified by Ni-NTA affinity chromatography,ULPI digestion,second Ni-NTA affinity chromatography and gel filtration chromatography.High-quality crystals were obtained in optimal conditions (0.1 mol/L HEPES pH 7,12% PEG 6000,0.2 mol/L MgCl₂,8 mmol/L CHAPS).The diffraction data of crystals were collected at BL18U1 beamline of Shanghai Synchrotron Radiation Facility(SSRF).The crystal structure of DehDIV-R with a resolution of 2.35? was successfully resolved by Molecular Replacement(MR).The Ramachandran plot shows that 98.02% of the amino acids are in the optimum region,indicating the rationality of the structure.The purification,crystallization and structural analysis of the DehDIV-R have laid a foundation for further understanding the relationship between structure and function.

Key words： R-2-haloacid dehalogenase Purification Crystallization X-ray diffraction

Received: 09 March 2018 Published: 11 September 2018

ZTFLH:

Q814

Corresponding Authors: Gang XU E-mail: xugang_1030@zju.edu.cn

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	Chao-di TONG
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Cite this article:

Chao-di TONG,Jian-ping WU,Li-rong YANG,Gang XU. Crystal Structural Analysis of DehDIV-R by X-ray Crystallography. China Biotechnology, 2018, 38(8): 19-25.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180803 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I8/19

Fig.1 SDS-PAGE analysis of first Ni-NTA affinity chromatography purification for DehDIV-R M:Marker;1:Supernatant;2:Flowthrough;3:Buffer A eluate;4:10% Buffer B eluate;5,6:30% Buffer B eluate;7-9:Buffer B eluate

Fig.2 SDS PAGE analysis of second Ni-NTA affinity chromatography purification for DehDIV-R after digested by ULP1 M:Marker;1:Before digested by ULP1;2:After digested by ULP1;3,4: Flowthrough;5,6:10% Buffer B eluate;7:Buffer B eluate

Fig.3 The gel filtration spectrum of DehDIV-R

Fig.4 SDS-PAGE analysis of gel filtration chromatography purification for DehDIV-R M:Marker;1-9:Fractions A71~A87

Fig.5 The crystal screening of DehDIV-R (a) HR2-144 H8 (b) PEGIIs H10 (c) PACT C10

Fig.6 The crystal of DehDIV-R after optimization

Table 1 The diffraction statistics of DehDIV-R crystal

Fig.7 X-ray diffraction pattern of the DehDIV-R crystal

Table 2 The refinement statistics of DehDIV-R crystal structure

Fig.8 The Ramachandran plot of DehDIV-R

Fig.9 The overall structure(a) and active pocket(b) of DehDIV-R


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