摘要
Ⅲ型聚酮合酶是以合成聚酮类化合物为主的一类重要生物合成酶。本文利用逆转录聚合酶链反应从中草药千层塔新鲜嫩叶中扩增聚酮合酶基因,得到一个Ⅲ型聚酮合酶全长cDNA。该基因全长1212 bp,编码404个氨基酸。与已知的其他植物来源的聚酮合酶氨基酸序列有约50%～66%的同源性。cDNA经双酶切后克隆至pQE81L,并导入大肠杆菌(E.coli)M15中表达,产生大量带寡聚组氨酸标记的重组酶,重组酶分子质量大小约为46.4 kDa。酶活性鉴定研究表明,该重组酶可催化芳香族底物、脂肪族底物生成系列非天然聚酮产物,尤其是其可催化N-甲基邻氨基苯甲酰CoA和丙二酰CoA生成1,3-二羟基-N-甲基-吖啶酮,吖啶酮生物碱一直被认为只能由吖啶酮合酶合成。该工作为研究千层塔中Ⅲ型聚酮合酶在天然药物石杉碱甲生物合成中的作用奠定基础。
A cDNA encoding novel type Ⅲ polyketide synthase（PKS） was cloned and sequenced from young leaves of Chinese club moss Huperzia serrata（Thunb.） Trev.by RT-PCR using degenerated primers based on the conserved sequences of known CHSs,and named as H.serrata PKS2.The terminal sequences of cDNA were obtained by the 3＇-and 5＇-RACE method.The full-length cDNA of H.serrata PKS2 contained a 1 212 bp open reading frame encoding a 46.4 kDa protein with 404 amino acids.The deduced amino acid sequence of H.serrata PKS2 showed 50 %-66% identities to those of other chalcone synthase super family enzymes of plant origin.The recombinant H.serrata PKS2 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus and showed unusually versatile catalytic potency to produce various aromatic tetraketides,including chalcones,benzophenones,phloroglucinols,and acridones.In particular,the enzyme accepted bulky starter substrates N-methylanthraniloyl-CoA,and carried out three condensations with malonyl-CoA to produce 1,3-dihydroxy-N-methylacridone.Interestingly,H.serrata PKS2 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens（Rutaceae）.
出处
《药学学报》
CAS
CSCD
北大核心
2011年第10期1273-1278,共6页
Acta Pharmaceutica Sinica
基金
浙江省自然科学基金资助项目(Y208631)
浙江省卫生厅项目(2008A003)
关键词
千层塔
Ⅲ型聚酮合酶
基因克隆
Huperzia serrata
type Ⅲ polyketide synthase
gene expression
