Characterisation of the First Enzymes Committed to Lysine Biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.在植物中，赖氨酸生物合成途径是除草剂开发与提升作物营养价值的理想靶点——鉴于赖氨酸是谷类作物中的限制性氨基酸。二氢吡啶二羧酸合酶（dihydrodipicolinate synthase, DHDPS）与二氢吡啶二羧酸还原酶（dihydrodipicolinate reductase, DHDPR）催化赖氨酸生物合成的前两个关键不可逆步骤。本研究首次对拟南芥（Arabidopsis thaliana）来源的DHDPS与DHDPR的结构与活性开展全面表征。拟南芥DHDPS酶（At-DHDPS2）的活性与细菌来源的该酶相近，但被（S）-赖氨酸的别构抑制作用更强。对At-DHDPS2的结构研究显示，（S）-赖氨酸结合于两个单体之间的裂隙处，明确了别构位点；但与既往研究不同的是，该结合并未伴随构象变化，提示结合可能引发蛋白质动力学改变而非大规模构象重排。拟南芥来源的DHDPR（At-DHDPR2）在催化过程中对NADH与NADPH具有相近的底物特异性，且对底物的结合亲和力较既往报道更强。尽管所有已知的细菌DHDPR酶均为四聚体结构，但分析超速离心与散射实验数据明确表明，At-DHDPR2在溶液中以二聚体形式存在。该二聚体蛋白的精确组装方式目前尚不明确，但基于X射线散射数据的从头建模结果与溶液中呈伸长结构的假说相符，而该结构与其他物种来源DHDPR的X射线晶体结构中观测到的所有可能二聚体配对方式均不匹配。对植物赖氨酸生物合成酶的结构与功能的这些新增认知，将为未来旨在提升作物初级生产的研究提供助力。