Deletion of a Single Amino Acid Residue Val337 from Rice (Oryza sativa) Os4CL5 Results in the Generation of New Substrate Specificities Toward Sinapate
Abstract：4-coumarate: coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. The catalytic activity of 4CL towards sinapate got a lot of attention at present. Only Arabidopsis thaliana At4CL4 and soybean (Glycine max) Gm4CL1 have catalytic ability toward sinapate so far. There is a conserved valine which is located in the substrate binding pocket by sequence comparison of rice (Oryza sativa subsp. japonica) Os4CL5 with At4CL4 and Gm4CL1. The existence of valine between Pro336 and Leu338 may eliminate the activity of Os4CL5 towards sinapate. In this paper, the expression vectors of wild-type Os4CL5 and mutant Os4CL5 with the deletion of Val337 were constructed. Then the recombinant plasmids were transformed into Escherichia coli BL21(DE3) respectively. The recombinant cells were grown and induced by isopropyl-β-d-thiogalactoside (IPTG). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the induced protein was about 57.0 kD consistent with the predicted value. After purification by affinity chromatography, the enzymatic properties of wild-type Os4CL5 and mutant Os4CL5 were primarily characterized. The results showed that wild-type Os4CL5 had different enzymatic activity towards the different substrate. The rank in order of turnover rate for different substrate was coumarate＞ferulate＞caffeate. It should be noted that sinapate was not accepted as a substrate under experimental conditions. The deletion of Val337 from rice Os4CL5 results in increased activity towards different substrate and the generation of new substrate specificities towards sinapate. This study provides a theoretical basis for the regulation of lignin biosynthesis by genetic engineering.
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