科微学术

生物工程学报

2025年4月6日 1:35 星期日
首页 > 过刊浏览>2024年第40卷第9期 >2786-2796. DOI:10.13345/j.cjb.240117
PDF HTML阅读 XML下载 导出引用 引用提醒
多功能氧化酶莨菪碱6β-羟化酶的研究进展
作者:
基金项目:

国家重点研发计划(2021YFC2102000)


Research progress of the multifunctional oxidase scopolamine 6β-hydroxylase
Author:
  • 摘要
    • | |
  • 访问统计
    • |
  • 参考文献 [46]
    • |
  • 相似文献
    • |
  • 引证文献
    • | |
  • 文章评论
    摘要:

    2-酮戊二酸/Fe2+依赖的双加氧酶能够实现复杂结构化合物sp3杂化C-H键的官能化反应，并且反应条件温和，对底物具有高度的区域和立体选择性。莨菪碱6β-羟化酶(hyoscyamine 6β-hydroxylase, H6H)属于此类双加氧酶，是催化合成东莨菪碱的最后两步的关键酶，能够依次实现莨菪碱的6β-羟化和6,7位的环氧化反应。本文介绍了莨菪碱6β-羟化酶催化机制、底物谱和应用进展，对该酶转化不同结构特点底物的羟化、环氧化等反应的潜在能力进行了评估，为后续对酶的设计改造和应用研究提供理论基础。

    Abstract:

    2-ketoglutarate (2-KG)/Fe2+-dependent dioxygenases can catalyze the highly specific regio- and stereoselective functionalization of C(sp3)-H bond of complex compounds under mild reaction conditions. Hyoscyamine 6β-hydroxylase (H6H), a member of these dioxygenases, catalyzes two consecutive oxidation reactions in the synthesis of scopolamine. The first reaction is the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine and the second is epoxidation of 6β-hydroxyhyoscyamine. This paper introduces the catalytic mechanism, substrate scope, and application of H6H and evaluates the possibility of this enzyme as a biocatalyst for the functionalization of C(sp3)-H bond in complex compounds with different structural characteristics via hydroxylation or epoxidation, providing a theoretical basis for modification and application of this enzyme.

    参考文献
    [1] SCHNEPEL C, SEWALD N. Enzymatic halogenation: timely strategy for regioselective C-H activation[J]. Chemistry, 2017, 23(50): 12064-12086.
    [2] BÜCHLER J, PAPADOPOULOU A, BULLER R. Recent advances in flavin-dependent halogenase biocatalysis: sourcing, engineering, and application[J]. Catalysts, 2019, 9(12): 1030.
    [3] ROMERO E, JONES BS, HOGG BN, RUÉCASAMAJO A, HAYES MA, FLITSCH SL, TURNER NJ, SCHNEPEL C. Enzymatic late-stage modifications: better late than never[J]. Angewandte Chemie (International Edition in English), 2021, 60(31): 16824-16855.
    [4] WU S, SNAJDROVA R, MOORE JC, BALDENIUS K, BORNSCHEUER UT. Biocatalysis: enzymatic synthesis for industrial applications[J]. Angewandte Chemie (International Edition in English), 2021, 60(1): 88-119.
    [5] DAVIDI D, LONGO LM, JABŁOŃSKA J, MILO R, TAWFIK DS. A bird’s-eye view of enzyme evolution: chemical, physicochemical, and physiological considerations[J]. Chemical Reviews, 2018, 118(18): 8786-8797.
    [6] REETZ MT. Laboratory evolution of stereoselective enzymes: prolific source of catalysts for asymmetric reactions[J]. Angewandte Chemie (International Edition in English), 2011, 50(1): 138-174.
    [7] STROHMEIER GA, PICHLER H, MAY O, GRUBER-KHADJAWI M. Application of designed enzymes in organic synthesis[J]. Chemical Reviews, 2011, 111(7): 4141-4164.
    [8] QU G, LI A, ACEVEDO-ROCHA CG, SUN ZT, REETZ MT. The crucial role of methodology development in directed evolution of selective enzymes[J]. Angewandte Chemie (International Edition in English), 2020, 59(32): 13204-13231.
    [9] ROCHA RA, SPEIGHT RE, SCOTT C. Engineering enzyme properties for improved biocatalytic processes in batch and continuous flow[J]. Organic Process Research & Development, 2022, 26(7): 1914-1924.
    [10] WOJDYLA Z, BOROWSKI T. Properties of the reactants and their interactions within and with the enzyme binding cavity determine reaction selectivities. The case of Fe(II)/2-oxoglutarate dependent enzymes[J]. Chemistry, 2022, 28(18): e202104106.
    [11] HASHIMOTO T, YAMADA Y. Hyoscyamine 6β-hydroxylase, 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures[J]. Plant Physiology, 1986, 81(2): 619-625.
    [12] LIU T, ZHU P, CHENG KD, MENG C, HE HX. Molecular cloning, expression and characterization of hyoscyamine 6β-hydroxylase from hairy roots of Anisodus tanguticus[J]. Planta Medica, 2005, 71(3): 249-253.
    [13] EL JABER-VAZDEKIS N, GONZÁLEZ C, RAVELO AG, ZÁRATE R. Cloning, characterization and analysis of expression profiles of cDNA encoding hyoscyamine 6beta-hydroxylase (H6H) from Atropa baetica Willk[J]. Plant Physiology and Biochemistry: PPB, 2009, 47(1): 20-25.
    [14] LI J, van BELKUM MJ, VEDERAS JC. Functional characterization of recombinant hyoscyamine 6β-hydroxylase from Atropa belladonna[J]. Bioorganic & Medicinal Chemistry, 2012, 20(14): 4356-4363.
    [15] PRAMOD KK, SINGH S, JAYABASKARAN C. Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L.[J]. Plant Physiology and Biochemistry: PPB, 2010, 48(12): 966-970.
    [16] FISCHER C, KWON M, RO D-K, van BELKUM MJ, VEDERAS JC. Isolation, expression and biochemical characterization of recombinant hyoscyamine-6β- hydroxylase from Brugmansia sanguinea-tuning the scopolamine production[J]. MedChemComm, 2018, 9(5): 888-892.
    [17] NAIK T, VANITHA SC, RAJVANSHI PK, CHANDRIKA M, KAMALRAJ S, JAYABASKARAN C. Novel microbial sources of tropane alkaloids: first report of production by endophytic fungi isolated from Datura metel L.[J]. Current Microbiology, 2018, 75(2): 206-212.
    [18] TIMMINS A, de VISSER SP. A comparative review on the catalytic mechanism of nonheme iron hydroxylases and halogenases[J]. Catalysts, 2018, 8(8): 314.
    [19] KLUZA A, WOJDYLA Z, MRUGALA B, KURPIEWSKA K, POREBSKI PJ, NIEDZIALKOWSKA E, MINOR W, WEISS MS, BOROWSKI T. Regioselectivity of hyoscyamine 6β-hydroxylase- catalysed hydroxylation as revealed by high-resolution structural information and QM/MM calculations[J]. Dalton Transactions, 2020, 49(14): 4454-4469.
    [20] JUMPER J, EVANS R, PRITZEL A, GREEN T, FIGURNOV M, RONNEBERGER O, TUNYASUVUNAKOOL K, BATES R, ŽÍDEK A, POTAPENKO A, BRIDGLAND A, MEYER C, KOHL SAA, BALLARD AJ, COWIE A, ROMERA-PAREDES B, NIKOLOV S, JAIN R, ADLER J, BACK T, et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596(7873): 583-589.
    [21] WAGNER JR, SØRENSEN J, HENSLEY N, WONG C, ZHU C, PERISON T, AMARO RE. POVME 3.0: software for mapping binding pocket flexibility[J]. Journal of Chemical Theory and Computation, 2017, 13(9): 4584-4592.
    [22] ZHU Q, LIU X. Characterization of non-heme iron aliphatic halogenase WelO5* from Hapalosiphon welwitschiiIC-52-3: identification of minimal protein sequence motif that confers enzymatic chlorination specificity in the biosynthesis of welwitindolelinones[J]. Beilstein Journal of Organic Chemistry, 2017, 13: 1168-1173.
    [23] HASHIMOTO T, KOHNO J, YAMADA Y. Epoxidation in vivo of hyoscyamine to scopolamine does not involve dehydration step[J]. Plant Physiology, 1987, 84(1): 144-147.
    [24] HASHIMOTO T, YAMADA Y. Purification and characterization of hyoscyamine 6β-hydroxylase from root cultures of Hyoscyamus niger L. hydroxylase and epoxidase activities in the enzyme preparation[J]. European Journal of Biochemistry, 1987, 164(2): 277-285.
    [25] USHIMARU R, RUSZCZYCKY MW, LIU HW. Changes in regioselectivity of H atom abstraction during the hydroxylation and cyclization reactions catalyzed by hyoscyamine 6β-hydroxylase[J]. Journal of the American Chemical Society, 2019, 141(2): 1062-1066.
    [26] MARTINEZ S, HAUSINGER RP. Catalytic mechanisms of Fe(II)- and 2-oxoglutarate-dependent oxygenases[J]. The Journal of Biological Chemistry, 2015, 290(34): 20702-20711.
    [27] SHIBASAKI T, SAKURAI W, HASEGAWA A, UOSAKI Y, MORI H, YOSHIDA M, OZAKI A. Substrate selectivities of proline hydroxylases[J]. Tetrahedron Letters, 1999, 40(28): 5227-5230.
    [28] KLEIN C, HÜTTEL W. A simple procedure for selective hydroxylation of L-proline and L-pipecolic acid with recombinantly expressed proline hydroxylases[J]. Advanced Synthesis & Catalysis, 2011, 353(8): 1375-1383.
    [29] SMIRNOV SV, SOKOLOV PM, KODERA T, SUGIYAMA M, HIBI M, SHIMIZU S, YOKOZEKI K, OGAWA J. A novel family of bactgenes[J]. Physiologia Plantarum, 2011, 143(4): 309-315.
    [47] MINOIA JM, VILLANUEVA ME, COPELLO GJ, RODRÍGUEZ TALOU J, CARDILLO AB. Recycling of hyoscyamine 6β-hydroxylase for the in vitro production of anisodamine an? scopolamine[J]. Applied Microbiology and Biotechnology, 2023, 107(11): 3459-3478.sis: from structural insights into practical applications[J]. Systems Microbiology and Biomanufacturing, 2021, 1(3): 275-290.
    [31] ZWICK CR III, RENATA H. Overview of amino acid modifications by iron- and α-ketoglutarate-dependent enzymes[J]. ACS Catalysis, 2023, 13(7): 4853-4865.
    [32] PAPADOPOULOU A, MEYER F, BULLER RM. Engineering Fe(II)/αketoglutarate-dependent halogenases and desaturases[J]. Biochemistry, 2023, 62(2): 229-240.
    [33] NEUGEBAUER ME, SUMIDA KH, PELTON JG, McMURRY JL, MARCHAND JA, CHANG MCY. A family of radical halogenases for the engineering of amino-acid-based products[J]. Nature Chemical Biology, 2019, 15: 1009-1016.
    [34] USHIMARU R, RUSZCZYCKY MW, CHANG WC, YAN F, LIU YN, LIU HW. Substrate conformation correlates with the outcome of hyoscyamine 6β-hydroxylase catalyzed oxidation reactions[J]. Journal of the American Chemical Society, 2018, 140(24): 7433-7436.
    [35] CAO YD, HE YC, LI H, KAI GY, XU JH, YU HL. Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing S14P/K97A mutant of hyoscyamine 6β-hydroxylase AaH6H[J]. Journal of Biotechnology, 2015, 211: 123-129.
    [36] CARDILLO AB, PERASSOLO M, SARTUQUI M, RODRÍGUEZ TALOU J, GIULIETTI AM. Production of tropane alkaloids by biotransformation using recombinant Escherichia coli whole cells[J]. Biochemical Engineering Journal, 2017, 125: 180-189.
    [37] YUN DJ, HASHIMOTO T, YAMADA Y. Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition[J]. Proceedings of the National Academy of Sciences of the United States of America, 1992, 89(24): 11799-11803.
    [38] HASHIMOTO T, MATSUDA J, YAMADA Y. Two-step epoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6β-hydroxylase[J]. FEBS Letters, 1993, 329(1/2): 35-39.
    [39] HÁKKINEN ST, MOYANO E, CUSIDÓ RM, PALAZÓN J, PIÑOL MT, OKSMAN-CALDENTEY KM. Enhanced secretion of tropane alkaloids in Nicotiana tabacum hairy roots expressing heterologous hyoscyamine-6β-hydroxylase[J]. Journal of Experimental Botany, 2005, 56(420): 2611-2618.
    [40] CARDILLO AB, TALOU JR, GIULIETTI AM. Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst[J]. Microbial Cell Factories, 2008, 7: 17.
    [41] LUDWIG-MÜLLER J, JAHN L, LIPPERT A, PÜSCHEL J, WALTER A. Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: strategies and applications[J]. Biotechnology Advances, 2014, 32(6): 1168-1179.
    [42] HASHIMOTO T, YUN DJ, YAMADA Y. Production of tropane alkaloids in genetically engineered root cultures[J]. Phytochemistry, 1993, 32(3): 713-718.
    [43] JOUHIKAINEN K, LINDGREN L, JOKELAINEN T, HILTUNEN R, TEERI TH, OKSMAN-CALDENTEY KM. Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering[J]. Planta, 1999, 208(4): 545-551.
    [44] KANG YM, LEE OS, JUNG HY, KANG SM, LEE BH, KARIGAR C, PRASAD T, BAHK JD, CHOI MS. Overexpression of hyoscyamine 6β-hydroxylase (h6h) gene and enhanced production of tropane alkaloids in Scopolia parviflora hairy root lines[J]. Journal of Microbiology and Biotechnology, 2005, 15(1): 91-98.
    [45] ZÁRATE R, EL JABER-VAZDEKIS N, MEDINA B, RAVELO ÁG. Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropa baetica by over-expressing the gene encoding hyoscyamine 6β-hydroxylase[J]. Biotechnology Letters, 2006, 28(16): 1271-1277.
    [46] WANG X, CHEN M, YANG C, LIU X, ZHANG L, LAN X, TANG K, LIAO Z. Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h
    相似文献
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

陈曦,吴洽庆,朱敦明. 多功能氧化酶莨菪碱6β-羟化酶的研究进展[J]. 生物工程学报, 2024, 40(9): 2786-2796

复制
分享
文章指标
  • 点击次数:294
  • 下载次数: 680
  • HTML阅读次数: 532
  • 引用次数: 0
历史
  • 收稿日期:2024-02-18
  • 最后修改日期:2024-05-29
  • 在线发布日期: 2024-09-24
  • 出版日期: 2024-09-25
文章二维码
您是第5998993位访问者
生物工程学报 ® 2025 版权所有

通信地址：中国科学院微生物研究所    邮编：100101

电话：010-64807509   E-mail：cjb@im.ac.cn

技术支持：北京勤云科技发展有限公司