Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum
Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering.
Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS; EC 188.8.131.52) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a KM (pyruvate) of 0.45 mM, KM (l-aspartate-4-semialdehyde) of 0.07 mM, kcat of 56 s−1, and is inhibited by lysine (IC 50 LYS of 0.033 mM), which agree well with previous studies using labor-intensive purification from wheat suspension cultures. We subsequently employed circular dichroism spectroscopy, analytical ultracentrifugation and small angle X-ray scattering to show that the recombinant enzyme is folded with 60% α/β structure and exists as a 7.5 S tetrameric species with a Rg of 33 Å and Dmax of 118 Å. This study is the first to report the biophysical properties of the recombinant Ta-DHDPS in aqueous solution and offers an excellent platform for future studies aimed at improving nutritional value and primary production of bread wheat.
KeywordsCD spectroscopy Class I aldolase Enzyme kinetics SAXS Sedimentation Wheat
TPSC acknowledges the National Health and Medical Research Council of Australia for fellowship support (APP1091976) and MAP the Australian Research Council for funding support (DP150103313). We would like to acknowledge the La Trobe University-Comprehensive Proteomics Platform, La Trobe University, Melbourne, Australia for providing the infrastructure and expertise. We would also like to acknowledge the support and assistance of the beamline scientists at the Australian Synchrotron, Victoria, Australia. Finally, we thank all members of the Perugini laboratory for helpful discussions during the preparation of this manuscript.
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Conflict of interest
There are no competing interests to declare.
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