红花石蒜苯丙氨酸解氨酶催化合成N-甲基-L-苯丙氨酸

Biocatalytic synthesis of N-methyl-L-phenylalanine by phenylalanine ammonia lyase from galanthamine-producing Lycoris radiata

【背景】N-甲基-L-苯丙氨酸是一种N-烷基化芳香氨基酸,是重要的手性合成单元/中间体/组成成分,在医药、农业、食品等领域有重要应用价值的代谢产物中广泛存在。N-烷基化芳香氨基酸的合成与制备仍具有巨大的挑战。【目的】在研究加兰他敏的生物合成过程中,我们从产加兰他敏的红花石蒜中克隆并表征苯丙氨酸解氨酶LrPAL3。LrPAL3催化区域及对映选择性的氢胺化反应得到L-苯丙氨酸。通过生物信息学分析,推测LrPAL3可能催化反式-肉桂酸的一步N-甲基胺化反应得到N-甲基-L-苯丙氨酸。【方法】将反式-肉桂酸与甲胺,以及表达LrPAL3的大肠杆菌全细胞一起孵育。HPLC-DAD及HRESIMS分析表明,上述反应产物为N-甲基-苯丙氨酸。为确定该产物的立体构型,将上述催化反应放大,通过分离纯化得到该酶催化反应产物。【结果】该化合物的氢谱数据及比旋光数据与N-甲基-L-苯丙氨酸标准品的数据一致。由此说明,LrPAL3能够催化反式-肉桂酸和甲胺发生N-烷基胺化反应,区域和立体专一性地生成N-甲基-L-苯丙氨酸。【结论】本研究为手性N-烷基氨基酸的不对称合成提供了一种全新的绿色、高效生物催化剂。通过对LrPAL3的蛋白质定向进化及代谢工程,将会进一步扩展LrPAL3的催化反应范围,以多种N-烷基胺类及取代的苯基丙烯酸为底物,实现手性N-烷基-芳基氨基酸的高效区域及立体选择性生物合成。

[Background]N-Methyl-L-phenylalanine,an N-alkylated aromatic amino acid,is a valuable chiral building block/intermediate/ingredient presented in many specialized metabolites that are very important in pharmaceutical,nutraceutical,and agrochemical industries.The synthesis and preparation of N-alkylated aromatic amino acids from aromaticα,β-unsaturated carboxylic acid remain challenging.[Objective]Herein we report a one-step biologically catalyzed N-methylamination of trans-cinnamic acid by LrPAL3,a phenylalanine ammonia lyase(PAL)from galanthamine-producing Lycoris radiata,to generate N-methyl-L-phenylalanine.[Methods]HPLC-DAD and HRESIMS analyses revealed that N-methyl-phenylalanine was produced when incubated trans-cinnamic acid and methylamine using the whole Escherichia coli BL21(DE3)cells expressing LrPAL3 as catalyst.[Results]The 1 H-NMR data and optical rotation of the enzymatic bioconversion product are in agreement with those of the authentic N-methyl-L-phenylalanine,which demonstrated the LrPAL3-catalyzed one-step regio-and enantioselective N-methylamination product of trans-cinnamic acid is N-methyl-L-phenylalanine.[Conclusion]This work provides an alternative biocatalyst for the asymmetric synthesis of valuable chiral N-methyl-L-phenylalanine.It paves a way to biologically synthesize N-alkylated amino acids through metabolic engineering and direct protein evolution of LrPAL3.