用于生物合成1,5-戊二胺的新的L-赖氨酸脱羧酶的突变研究

1,5-Diaminopentane, a nitrogenous base chemical compound with important biological activity in the living cells, and has also been widely applications in the fields of agriculture, pharmaceutics, and chemical industry, etc. L-Lysine decarboxylase (EC 4. 1. 1. 18), a key limited enzyme can catalyze the decarboxylation of L-lysine to form 1,5-diaminopentane, which enzymatic activity contribute significantly to the biosynthesis efficiency of 1,5-diaminopentane. It’s important to isolate the metagenome-derived L-lysine decarboxylase for the biosynthesis efficiency of 1,5-diaminopentane. Metagenomic technology is a short-cut approach to obtain new genes from uncultured microorganisms. In the previous study, a subtropical soil metagenomic library had been constructed, and a putative L-lysine decarboxylase gene named ldc1E had been isolated using functional and sequence-based screening strategy. The derived amino acid sequence comparison and structure analysis indicated that Ldc1E and other putative L-lysine decarboxylases were moderate related. The ldc1E gene was highly expressed in Escherichia coli competent cells and the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the recombinant Ldc1E protein had been performed. The final results revealed that Ldc1E protein had higher enzyme specific activity, 5'-PLP-independent, and its substrate profiles were distinctly compared to the previous reported of L-lysine decarboxylases. In this application, the structure and function research of Ldc1E protein will be further focused. At first, the 3D structure will be completed using the homology modeling analysis program. Subsequently, the identification prediction of the key differential conserved amino acids will be completed through multi-alignment analysis technology and molecular docking technology, etc. The functional characterization of the key differential amino acids information of Ldc1E protein related to enzymatic activity, coenzyme factors and substrate profiles will be demonstrated completely through DNA site-directed mutagenesis technology, respectively. Finally, the structure and the function of Ldc1E protein will be demonstrated directly. This application successfully will benefit to the development of theory references for the new molecular catalytic mechanisms of L-lysine decarboxylase for the biosynthesis of 1,5-diaminopentane.

1,5-戊二胺是一种具有生物活性的含氮碱，具有重要的生理调节作用，在农业、医药和化工等领域也具有广泛的用途。L-赖氨酸脱羧酶(EC 4.1.1.18)催化L-赖氨酸脱羧生成1,5-戊二胺，该酶的活性高低，直接制约了1,5-戊二胺的生物合成效率。因此分离新的L-赖氨酸脱羧酶解决合成体系中酶活力不高的瓶颈问题非常重要。本项目前期已完成了亚热带山区土壤宏基因组文库中新的L-赖氨酸脱羧酶Ldc1E的分离和功能研究，发现Ldc1E蛋白具备较高的酶活力、不完全依赖辅酶5'-PLP且作用底物谱广泛等新的功能特征。本项目拟利用同源建模和分子对接技术等完成Ldc1E蛋白关键差异氨基酸残基的预测，通过基因定点突变技术完成关键差异氨基酸残基的突变，完成突变酶的功能分析，完成关键差异氨基酸残基与酶学功能特征之间的关联性研究。本研究将对可能的新的L-赖氨酸脱羧酶作用机制提供理论支持，因而具有重要的实践意义。

L-赖氨酸脱羧酶 (EC 4.1.1.18)催化L-赖氨酸脱羧生成1,5-戊二胺，该酶的活性高低，直接制约了1,5-戊二胺的生物合成效率。因此分离新的L-赖氨酸脱羧酶解决合成体系中酶活力不高的瓶颈问题非常重要。本项目完成了亚热带山区土壤宏基因组文库中新的L-赖氨酸脱羧酶Ldc1E（GenBank登录号：KX463450）的分离和功能研究，新的L-赖氨酸脱羧酶基因ldc1E相关序列分析、功能域分析以及进化关系分析结果表明Ldc1E属于III型磷酸吡哆醛依赖型脱羧酶和天冬氨酸氨基转移酶超家族。同时发现Ldc1E蛋白具备较高的酶活力且作用底物谱广泛等新的功能特征。产物鉴定结果表明：Ldc1E具有催化L-赖氨酸转化为戊二胺的能力；其最适作用温度为40 °C，最适pH为6.5；Km值为1.08 mM，Vmax值为23.13 μM/min，kcat 值为5.09/s，比活力为1.53 U/mg；Ldc1E蛋白在30 °C条件下保温1 h，稳定性较好，酶活性保持在80%左右，在pH 6.5-7.0条件下保存24 h，稳定性较好，酶活性保持在80%左右；5 mM的金属离子Mg2+和Ca2+对Ldc1E有微弱的激活作用；Ldc1E对化学试剂EDTA和DMSO表现出良好的耐受性；底物谱分析结果表明，Ldc1E对L-精氨酸和L-鸟氨酸均具有一定的催化作用。基于序列分析、同源建模分析以及定点突变技术，对Ldc1E与辅因子、底物分子结合的关键位点及其作用机制进行研究，结果表明，Ldc1E在空间结构和活性中心具有一定的保守性。突变体Y308A、E526D、V524A和L536A的酶活大幅度降低，表明Y308、E526、V524和L536位点的突变对酶催化活性有显著影响。突变体K367A、K367R、S364A、H366A、E526A和W333A的酶活性完全丧失，表明K367、S364、H366、E526和W333就是Ldc1E的关键结合位点，对催化活性起着至关重要的作用。我们推测：K367、S364、H366和W333的侧链与辅因子PLP之间存在相互作用力，从而使Ldc1E不能与底物发生催化作用；E526、V524和L536的侧链与底物分子之间存在相互作用力，这3个位点的突变导致底物在活性位点处的空间构象不稳定。该项目系列研究成果丰富了L-赖氨酸脱羧酶资源库，而且为该酶的工业化应用奠定了基础。