Streptomyces aidingensis CGMCC 4.5739中环二肽氧化酶DmtD3_E3的体外生化功能  

In vitro biochemical function of cyclodipeptide oxidase DmtD3_E3 from Streptomyces aidingensis CGMCC 4.5739

作　　者：姜玥辰 姚婷婷[1] 袁伟程 李文利[1] JIANG Yuechen;YAO Tingting;YUAN Weicheng;LI Wenli(Key Laboratory of Marine Drugs of Ministry of Education,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,Shandong,China)

机构地区：[1]中国海洋大学医药学院海洋药物教育部重点实验室,山东青岛266003

出　　处：《微生物学通报》2022年第11期4608-4616,共9页Microbiology China

基　　金：国家自然科学基金(32070054,31900049,81991525)。

摘　　要：【背景】环二肽合酶(cyclodipeptide synthase,CDPS)途径中新颖后修饰酶的挖掘对获得结构新颖活性良好的二酮哌嗪类化合物具有重要意义。前期研究中发现来源于Streptomyces aidingensis CGMCC 4.5739的环二肽合酶基因簇dmt3中dmt A3B3C3可编码二酮哌嗪—萜类化合物drimentines(DMTs),推测其下游环二肽氧化酶基因dmt D3_E3也参与了DMTs的生物合成,但其功能一直未鉴定。【目的】对S.aidingensis CGMCC 4.5739中环二肽合酶基因簇dmt3内的环二肽氧化酶Dmt D3_E3的功能进行表征,为增加二酮哌嗪类化合物结构多样性提供功能元件。【方法】从S.aidingensis CGMCC 4.5739的基因组中克隆环二肽氧化酶基因dmt D3_E3,构建重组表达质粒p WLI209,并在大肠杆菌BL21(DE3)中可溶性表达。通过建立体外酶促反应,运用液质联用(high performance liquid chromatography-mass spectrometry,HPLC-MS)和核磁共振(nuclear magnetic resonance,NMR)等方法确定催化产物结构。【结果】环二肽氧化酶Dmt D3_E3可催化环二肽cyclo-(L-Trp-L-Leu)(c WL)的C14-C17位氧化脱氢形成cyclo-(L-Trp-L-ΔLeu)(c WΔL)。此外Dmt D3_E3还可以催化环二肽cyclo-(L-Trp-L-Ala)(c WA)的C10-C11位脱氢生成cyclo-(L-Trp-L-ΔAla)(cΔWA),具有底物宽泛性。【结论】本研究通过对环二肽合酶生物合成途径中新颖环二肽氧化酶的挖掘和表征,为后续通过组合生物合成及合成生物学手段生成“非天然”二酮哌嗪类化合物衍生物奠定了基础。[Background]The establishment of cyclodipeptide synthase(CDPS)-associated tailoring enzymes offers particular promise for the generation of diketopiperazines with novel structures and good bioactivities.In the previous studies,coded diketopiperazines of the dmtA3B3C3 in CDPS gene cluster-terpenoid drimentines(DMTs)was identified in Streptomyces aidingensis CGMCC 4.5739.It was speculated that dmtD3-E3 in the downstream of CDPS participated in the biosynthesis of DMTs.However,the function of dmtD3_E3 has not been characterized.[Objective]To characterize the cyclodipeptide oxidase dmtD3_E3 in the gene cluster dmt3 of CDPS in S.aidingensis CGMCC 4.5739,and provide functional elements for structural diversity study of diketopiperazines.[Methods]dmtD3_E3 was cloned from the genome of S.aidingensis CGMCC 4.5739,and the recombinant plasmid pWLI209 was constructed and expressed soluble in Escherichia coli BL21(DE3).In vitro enzymatic reactions were performed,and high performance liquid chromatography-mass spectrometry(HPLC-MS)and nuclear magnetic resonance(NMR)were used to ensure the structure of catalysate.[Results]The cyclodipeptide oxidase dmtD3_E3 catalyzed the oxidative dehydrogenation of C14-C17 in cyclo-(LTrp-L-Leu)(cWL)to generate cWΔL,and the dehydrogenation of C10-C11 in cyclo-(L-Trp-L-Ala)(cWA)to generate cΔWA.The results showed that dmtD3_E3 demonstrated broad substrate specificity.[Conclusion]This study explored and characterized the novel cyclodipeptide oxidase dmtD3_E3 in the CDPS biosynthesis pathway,laying a foundation for the further generation of“non-natural”diketopiperazines through strategies of combinatorial biosynthesis and synthetic biology.

关 键 词：环二肽氧化酶 Streptomyces aidingensis CGMCC 4.5739 二酮哌嗪类化合物 环二肽合酶生物合成途径 

分 类 号：Q78[生物学—分子生物学]