摘要
鼠李糖合成酶(rhamnose synthase,RHM)是尿苷二磷酸鼠李糖(UDP-Rha)生物合成的关键酶,其在还原型辅酶Ⅰ和还原型辅酶Ⅱ的存在下,可将尿苷二磷酸葡萄糖(UDP-Glc)转化为UDP-Rha。本文以酵母提取物(yeast extract,YE)诱导的欧洲花楸悬浮细胞为研究材料,基于前期转录组数据,筛选并克隆出2条RHM基因,命名为SaRHM1(GenBank登录号MK213340)和SaRHM2(GenBank登录号MK213341)。SaRHM1和SaRHM2基因的开放阅读框分别为2007、2040 bp,分别编码668、679个氨基酸,生物信息学分析推测其分子质量分别为75.25、76.26 kD,理论等电点(theoretical pI)分别为7.24、6.41,两者均具有RHM家族的保守结构域(GxxGxxG/A和YxxxK)。多重序列比对与系统进化树显示,SaRHM1和SaRHM2与其他物种的RHM具有较高的同源性。体外酶促反应显示,重组蛋白SaRHM1和SaRHM2均具有将UDP-Glc转化为UDP-Rha的功能。酶促动力学参数显示,SaRHM1和SaRHM2反应的最佳pH分别为9和8,温度均为40℃,其中SaRHM1和SaRHM2的Km值分别为212.4±56.70和361.0±63.74μmol·L^(-1),Vmax值分别为235.5±18.98和516.5±22.30 nmol·min^(-1)·μg^(-1)。本研究首次报道了欧洲花楸RHM基因,并验证了其功能,为后续天然产物鼠李糖苷的生物合成提供鼠李糖供体。
Rhamnose synthase(RHM)is a key enzyme in the biosynthesis of uridine diphosphate rhamnose(UDP-Rha),reversibly converting uridine diphosphate-glucose(UDP-Glc)into UDP-Rha in the presence of NADH or NADPH.In this research,yeast extract(YE)was used to stimulate Sorbus aucuparia suspension cells.Based on a previous study of the transcriptome database of S.aucuparia suspension cells,two RHMs were cloned from S.aucuparia and named SaRHM1(GenBank No.:MK213340)and SaRHM2(GenBank No.:MK213341).The SaRHM1 gene contained a 2007 bp open reading frame(ORF)encoding a polypeptide of 668 amino acids with a molecular weight of 75.25 kD,and a theoretical isoelectric point(pI)of 7.24.The SaRHM2 gene contained a 2040 bp ORF encoding a polypeptide of 679 amino acids with a molecular weight of 76.26 kD and pI of 6.41.Bioinformatic analysis indicated that SaRHM1 and SaRHM2 contained two special sequences of GxxGxxG/A and YxxxK.Multiple sequence alignments and phylogenetic trees show that SaRHM1 and SaRHM2 have high sequence similarity with other plant species of RHMs.The results of enzyme activity assays in vitro revealed that both recombinant SaRHM1 and SaRHM2 are able to convert UDP-Glc into UDP-Rha.SaRHMs displayed maximum activity at 40℃and a pH of 8 and 9,respectively.The Km values of SaRHM1 and SaRHM2 for UDPGlc were 212.4±56.70 and 361.0±63.74μmol·L^(-1),respectively,with Vmax values of 235.5±18.98 and 516.5±22.30 nmol·min^(-1)·μg^(-1),respectively.This study reports the cloning and sequencing of RHMs from S.aucuparia and verifies their function,which likely provide rhamnose donors for the subsequent biosynthesis of rhamnosides.
作者
周良云
李佳兴
杨健
王升
吕朝耕
郭兰萍
ZHOU Liang-yun;LI Jia-xing;YANG Jian;WANG Sheng;Lü Chao-geng;GUO Lan-ping(Comprehensive Experimental Station of Guangzhou,Chinese Materia Medica,China Agriculture Research System,School of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《药学学报》
CAS
CSCD
北大核心
2021年第1期328-335,共8页
Acta Pharmaceutica Sinica
基金
国家重点研发计划项目(2017YFC1700701)
国家自然科学基金项目(81703648,81603239,81891014)
广东省普通高校青年创新人才类项目(2018KQNCX133)。
