科微学术

微生物学通报

Streptomyces aidingensis CGMCC 4.5739中环二肽氧化酶DmtD3_E3的体外生化功能
作者:
基金项目:

国家自然科学基金(32070054, 31900049, 81991525)


In vitro biochemical function of cyclodipeptide oxidase dmtD3_E3[t1] from Streptomyces aidingensis CGMCC 4.5739
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [16]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    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dmt3dmtA3B3C3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dmtD3_E3也参与了DMTs的生物合成,但其功能一直未鉴定。【目的】对S.aidingensisCGMCC 4.5739中环二肽合酶基因簇dmt3内的环二肽氧化酶DmtD3_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.aidingensisCGMCC 4.5739的基因组中克隆环二肽氧化酶基因dmtD3_E3,构建重组表达质粒pWLI209,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DmtD3_E3可催化环二肽cyclo-(L-Trp-L-Leu) (cWL)的C14-C17位氧化脱氢形成cyclo-(L-Trp-L-ΔLeu) (cWΔL)。此外DmtD3_E3还可以催化环二肽cyclo-(L-Trp-L-Ala) (cWA)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。

    Abstract:

    [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- (L-Trp-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.

    参考文献
    [1] Gisin BF, Merrifield RB. Carboxyl-catalyzed intramolecular aminolysis. A side reaction in solid-phase peptide synthesis[J]. Journal of the American Chemical Society, 1972, 94(9): 3102-3106
    [2] Borthwick AD. 2,5-diketopiperazines: synthesis, reactions, medicinal chemistry, and bioactive natural products[J]. Chemical Reviews, 2012, 112(7): 3641-3716
    [3] Harken L, Li SM. Modifications of diketopiperazines assembled by cyclodipeptide synthases with cytochrome P450 enzymes[J]. Applied Microbiology and Biotechnology, 2021, 105(6): 2277-2285
    [4] Kohn H, Widger W. The molecular basis for the mode of action of bicyclomycin[J]. Current Drug Targets Infectious Disorders, 2005, 5(3): 273-295
    [5] Lautru S, Gondry M, Genet R, Pernodet JL. The albonoursin gene cluster of S. noursei: biosynthesis of diketopiperazine metabolites independent of nonribosomal peptide synthetases[J]. Chemistry & Biology, 2002, 9(12): 1355-1364
    [6] Sun CH, Luo ZY, Zhang WL, Tian WY, Peng HD, Lin Z, Deng ZX, Kobe B, Jia XY, Qu XD. Molecular basis of regio-and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines[J]. Nature Communications, 2020, 11: 6251
    [7] Harken L, Liu J, Kreuz O, Berger R, Li SM. Biosynthesis of guatrypmethine C implies two different oxidases for exo double bond installation at the diketopiperazine ring[J]. ACS Catalysis, 2022, 12(1): 648-654
    [8] Yao TT, Liu J, Liu ZZ, Li T, Li HY, Che Q, Zhu TJ, Li DH, Gu QQ, Li WL. Genome mining of cyclodipeptide synthases unravels unusual tRNA-dependent diketopiperazine-terpene biosynthetic machinery[J]. Nature Communications, 2018, 9: 4091
    [9] Yao TT, Liu J, Jin EJ, Liu ZZ, Li HY, Che Q, Zhu TJ, Li DH, Li WL. Expanding the structural diversity of drimentines by exploring the promiscuity of two N-methyltransferases[J]. iScience, 2020, 23(7): 101323
    [10] Gondry M, Lautru S, Fusai G, Meunier G, Ménez A, Genet R. Cyclic dipeptide oxidase from Streptomyces noursei. Isolation, purification and partial characterization of a novel, amino acyl alpha, beta-dehydrogenase[J]. European Journal of Biochemistry, 2001, 268(6): 1712-1721
    [11] Giessen TW, Von Tesmar AM, Marahiel MA. Insights into the generation of structural diversity in a tRNA-dependent pathway for highly modified bioactive cyclic dipeptides[J]. Chemistry & Biology, 2013, 20(6): 828-838
    [12] Liu J, Yang YL, Harken L, Li SM. Elucidation of the streptoazine biosynthetic pathway in Streptomyces aurantiacus reveals the presence of a promiscuous prenyltransferase/cyclase[J]. Journal of Natural Products, 2021, 84(12): 3100-3109
    [13] Shi J, Xu X, Zhao EJ, Zhang B, Li W, Zhao Y, Jiao RH, Tan RX, Ge HM. Genome mining and enzymatic total biosynthesis of purincyclamide[J]. Organic Letters, 2019, 21(17): 6825-6829
    [14] Le Chevalier F, Correia I, Matheron L, Babin M, Moutiez M, Canu N, Gondry M, Lequin O, Belin P. In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2, 5-diketopiperazines in Escherichia coli[J]. Microbial Cell Factories, 2020, 19(1): 178
    [15] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning[M]. Cold spring harbor laboratory press, New York, 1989: 1814-1875
    [16] Couladouros EA, Magos AD. Solid-phase total synthesis of (–)-phenylhistine and (–)-aurantiamine. Synthesis of a diverse dehydro-2, 5-diketopiperazine library. Part II[J]. Molecular Diversity, 2005, 9(1/2/3): 111-121
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

姜玥辰,姚婷婷,袁伟程,李文利. Streptomyces aidingensis CGMCC 4.5739中环二肽氧化酶DmtD3_E3的体外生化功能[J]. 微生物学通报, 2022, 49(11): 4608-4616

复制
分享
文章指标
  • 点击次数:304
  • 下载次数: 958
  • HTML阅读次数: 1229
  • 引用次数: 0
历史
  • 收稿日期:2022-03-22
  • 最后修改日期:2022-04-19
  • 录用日期:2022-04-19
  • 在线发布日期: 2022-11-07
  • 出版日期: 2022-11-20
文章二维码