全霉素生物合成途径中的非核糖体肽合成酶HlmE的功能研究

Function study of nonribosomal peptide synthetase HlmE from biosynthetic pathway of holomycin

目的:非核糖体肽合成酶(nonribosomal peptide synthetase,NRPS)是二硫吡咯酮类化合物骨架合成的关键,本研究对全霉素(holomycin)生物合成途径中的NRPS HlmE进行酶学功能研究,以期为二硫吡咯酮类化合物生物合成途径的解析奠定基础.方法:利用原核表达系统表达NRPS HlmE,并通过亲和层析方法和快速蛋白质液相层析(fast protein liquid chromatography,FPLC)纯化仪器获得目的蛋白;利用高效液相色谱法(high performance liquid chromatography,HPLC)和基质辅助激光解吸电离-飞行时间质谱法(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)对HlmE的功能进行研究.结果:本研究成功在体外纯化得到高纯度HlmE,HPLC检测到了腺嘌呤核苷单磷酸(adenosine monophosphate,AMP)和半胱氨酰-AMP的产生,证明了HlmE中腺苷化(adenylation,A)结构域的活性;MALDI-TOF MS法证明HlmE具有挂载底物半胱氨酸的功能.结论:本研究纯化得到的NRPS HlmE可以活化并挂载底物半胱氨酸,为进一步研究NRPS的功能以及深入研究二硫吡咯酮类化合物的生物合成途径奠定基础.

Objective:Nonribosomal peptide synthetase(NRPS)is critical for the biosynthesis of dithiolopyrrolone compounds.In this paper,the enzymatic function of the NRPS HlmE in the biosynthetic pathway of holomycin was studied,and it was expected to pave a foundation for uncovering the biosynthetic pathway of dithiolopyrrolone natural products.Methods:The NRPS HlmE was expressed in prokaryotic expression system and the target protein was purified through affinity chromatography method in the fast protein liquid chromatography(FPLC)purification system.Based on the application of high performance liquid chromatography(HPLC)and matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF MS),the function of HlmE was explored.Results:HlmE was purified with high purity in vitro,adenosine monophosphate(AMP)and cysteine-AMP were both detected by HPLC,which proved the bio-activity of the adenylation(A)domain of HlmE.The results of MALDI-TOF MS demonstrated that HlmE could carry the substrate cysteine.Conclusion:The purified NRPS HlmE in this paper could activate and carry cysteine,which paved a foundation for further investigating the function of NRPS and the biosynthetic pathway of dithiolopyrrolone compounds.