Abstract
The rat aryl sulfotransferases IV (AST IV) has been used to catalyze 3′-phosphoadenosine-5′-phosphate (PAP) into the sulfuryl group donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in biotechnological production of glycosaminoglycans. The performance of native AST IV is not satisfying due to the lower catalytic activity with substrate PAP. In the present study, we achieved secretory expression of the AST IV and improved its catalytic efficiency by molecular engineering. Fusion with signal peptides Cex, YebF and PelB allow for secretory expression of AST IV and the secreted AST IV yield reached 4.21 ± 0.23 U/mL, 8.67 ± 0.34 U/mL and 21.35 ± 0.87 U/mL, respectively. Modification of PelB further increased the secretory expression by more than fourfold, to 89.67 ± 1.34 U/mL. On this basis, molecular evolution of the predicted PAP-binding pocket gate loop was performed and a positive mutant L89S/E90L with higher activity was identified. Considering the importance of the sites Leu89 and Glu90, we performed site-saturation mutagenesis and found the mutant L89M/E90Q with much higher PAP affinity (Km= 0.46 ± 0.02 mM) and catalytic efficiency (kcat/Km = 1816.32 ± 12.72/s/M). The secretory expression of the AST IV variant L89M/E90Q with higher catalytic efficiency should facilitate the studies on biosynthesis of sulfated polysaccharides.
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Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (31670092), the Fundamental Research Funds for the Central Universities (JUSRP51707A), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-16), and the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT_15R26) and the 111 Project.
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ZZ and ZK designed the experiments. QL, RX, and BW carried out all the experiments and analyzed data. GD wrote the manuscript.
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Zhou, Z., Li, Q., Xu, R. et al. Secretory expression of the rat aryl sulfotransferases IV with improved catalytic efficiency by molecular engineering. 3 Biotech 9, 246 (2019). https://doi.org/10.1007/s13205-019-1781-x
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DOI: https://doi.org/10.1007/s13205-019-1781-x