Positive Effects of Isopropanol as a Co-Precipitant in Glycerol-3-Phosphate Acyltransferase Crystallization
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Glycerol-3-phosphate acyltransferase (GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid (LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, the crystals were screened by hanging-drop and only clusters were obtained. The crystals were optimized by adjusting temperature, pH, protein concentration, or precipitant, but little improvement. To improve the interaction between protein and precipitant, the isopropanol was applied as co-precipitant. The qualified crystals formed. It’s suggested that isopropanol is critical to affect protein crystallization by altering polyethylene glycol (PEG)-water-protein interaction when PEG serves as precipitant. The resulting crystal diffracted to a resolution of 2.75 Å and belonged to space group P1, with unit-cell parameters a = 50.79, b = 80.09, c = 88.21 Å, and α = 62.85, β = 73.04, γ = 80.53°. This work introduced a new strategy to optimize the crystal of the heterogeneous catalysis enzymes like GPAT and provided the fundamental structural information for the oriented synthesis of marine microalgae glycerolipid.
Key wordsisopropanol polyethylene glycol (PEG) optimization glycerol-3-phosphate acyltransferase (GPAT) crystallization
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We appreciate the donation of LiGPAT plasmid by Professor Zhigang Zhou, College of Aqua-Life Science and Technology, Shanghai Ocean University, and thank the staff of beamline BL19U at the Shanghai Synchrotron Radiation Facility, People’s Republic of China for assistance in synchrotron X-ray data collection. This study was financially supported by the National Natural Science Foundation of China (Nos. 21576253, 31500294 and 31470432).
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