多克沙六烯、环氧体和阿拉奇酮酸的环氧代谢物酶合成
概要
我们提出了一种有用的方法,用于使用细菌细胞色素,对阿氏酸(AA)、多沙六烯酸(DHA)和乙二苯甲酸(EPA)的特异性环氧乙烷和环氧乙烷酶的环氧乙烷和环氧乙烷的再三氧化二氮进行纯化。P450 酶 (BM3)。
Abstract
各种多不饱和脂肪酸(PUFAs)的环氧树脂代谢物,称为环氧脂肪酸,在人体生理学中有着广泛的作用。这些代谢物是由细胞色素P450类酶内分泌产生的。由于其多样化和强大的生物效应,人们对研究这些代谢物产生了浓厚的兴趣。确定这些代谢物在体内的独特作用是一项艰巨的任务,因为环氧脂肪酸必须首先获得大量且纯度高。从天然来源获得化合物通常是劳动密集型的,可溶性环氧乙烷(sEH)会迅速水解代谢物。另一方面,通过化学反应获得这些代谢物的效率非常低,因为很难获得纯反热敏体和抗性剂,产量低,并且广泛(和昂贵的)纯化。在这里,我们提出一个有效的酶合成19(S),20 (R) – 和16 (S),17 (R)-环氧乙烷酸 (EDPs) 从DHA通过环氧化与BM3, 一种细菌CYP450酶从杆菌分离梅盖特(在大肠杆菌中很容易表达)。使用核磁共振光谱 (NMR)、高性能液相色谱 (HPLC) 和质谱 (MS) 进行纯度的特性和测定。本程序说明了PUFA环氧代谢物酶合成的好处,并适用于其他脂肪酸的环氧化,包括甲氧乙酸(AA)和乙酸苯甲酸(EPA),以生产类似的环氧乙烷酸酸(EETs)和四烯酸(EEQs)。
Introduction
近年来,随着人们对多不饱和脂肪酸(特别是欧米茄-3和欧米茄-6多不饱和脂肪酸)在人类生物学中的作用的兴趣与日俱增,研究人员已经注意到其代谢物具有广泛的吸引力。展览。特别是环氧脂肪酸代谢物产生的细胞色素P450类酶一直是人们关注的焦点。例如,许多PUFA环氧乙烷,包括环氧乙烷酸(EETs)、环氧二甲酸(EDPs)和环氧乙烷四甲酸(EEQs),在调节血压和炎症方面起着关键作用1,2,3,4,5.有趣的是,已知AA和EPA环氧树脂的特定异构体对血管收缩6、7具有不同影响。虽然EET和EEQ的内窥剂和再血管异构体的生理影响已经记录在案,但人们对DHA形成的类似环氧二苯丙酸(EDPs)的影响知之甚少。鱼油8的广泛使用,富含EPA和DHA,也激起了对EDPs9的兴趣。这些补充剂的好处被认为是部分由于下游DHA代谢物(16,17-EDP和19,20-EDP是最丰富的),因为在体内的EDP水平与膳食10中的DHA量协调得很好, 11.
通过代谢组学、化学生物学和其他方法研究这些环氧脂肪酸的机制和目标已被证明具有挑战性,部分原因在于它们作为再血管和立体异构体的混合物存在,以及一种获得纯量的需要反古体和再血管等位。事实证明,用化学合成这些化合物的传统方法是无效的。使用环氧树脂等环氧树脂有许多缺点,特别是缺乏环氧化选择性,这需要昂贵和费力地纯化单个再血管体和抗氧化剂。DHA和EPA代谢物的完全合成是可能的,但也存在缺点,使得大规模合成不切实际,如高成本和低产量12,13。有效的整体生产可以通过酶合成来实现,因为酶反应是regio-和立体选择性14。研究表明,AA和EPA的酶环氧化(与BM3)同时具有反选择性和内选选择性15,16,17,18,但这个程序没有测试与DHA,或在大型规模。我们该方法的总体目标是扩大和优化这种化学环氧化,以迅速产生大量的纯环氧脂肪酸作为各自的抗氧化剂。使用这里介绍的方法,研究人员可以获得一种简单且经济高效的策略来合成EDPs和其他PUFA环氧代谢物。
Protocol
Representative Results
Discussion
我们在这里提出了一种操作简单且经济高效的方法,用于制备DHA的两种最丰富的环氧代谢物 – 19,20 和 16,17-EDP。这些环氧脂肪酸可以使用野生型BM3酶以高抗纯(作为S,R-等体)形式制备。下面将介绍可用于故障排除的几个关键点,以及我们制备 AA 和 EPA 的环氧树脂代谢物的方法的扩展。
BM3 存储指南
在-78°C冷冻室储存之前,将蛋白质溶液与同等?…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作由R00 ES024806(国家卫生研究院)、DMS-1761320(国家科学基金会)和密歇根州立大学的启动基金资助。作者希望感谢杨俊博士(加州大学戴维斯分校)和拉利莎·卡查拉博士(密歇根州立大学)协助优化酶反应,以及托尼·席尔米勒博士(MSU质谱和代谢组学设施)协助HRMS数据采集。
Materials
| Ammonium Bicarbonate | Sigma | 9830 | NA |
| Ampicillin | GoldBio | A30125 | NA |
| Anhydrous magnesium sulfate | Fisher Scientific | M65-3 | NA |
| Anhydrous methanol | Sigma-Aldrich | 322515 | NA |
| Anhydrous sodium sulfate | Fisher Scientific | S421-500 | NA |
| Anhydrous toluene | Sigma-Aldrich | 244511 | NA |
| Arachidonic Acid (AA) | Nu-Chek Prep | U-71A | Air-sensitive. |
| Diethyl Ether | Sigma | 296082 | NA |
| DMSO (molecular biology grade) | Sigma-Aldrich | D8418 | NA |
| Docosahexaenoic Acid (DHA) | Nu-Chek Prep | U-84A | Air-sensitive. |
| EDTA (ethylenediaminetetraacetic acid) | Invitrogen | 15576028 | NA |
| Eicosapentaenoic Acid (EPA) | Nu-Chek Prep | U-99A | Air-sensitive. |
| Ethyl acetate | Sigma | 34858 | NA |
| Flash column cartridges 25, 40, 4, 12 g sizes | Fisher Scientific | 145170203, 145154064, 5170200 | Alternatively, conventional column chromatography can be used |
| Formic acid (HPLC Grade) | J.T. Baker | 0128-01 | NA |
| Glycerol | Sigma | G7757 | NA |
| Hexanes | VWR | BDH24575 | NA |
| LB Broth | Sigma | L3022 | NA |
| Lithium hydroxide | Sigma-Aldrich | 442410 | NA |
| Magnesium chloride | Fisher Scientific | 2444-01 | NA |
| Methanol (HPLC grade) | Sigma-Aldrich | 34860-41-R | NA |
| NADPH Tetrasodium Salt | Sigma-Aldrich | 481973 | Air-sensitive. |
| Oxalic acid | Sigma-Aldrich | 194131 | NA |
| pBS-BM3 transfected DH5α E. coli | NA | NA | NA |
| PMSF (phenylmethanesulfonyl fluoride) | Sigma | P7626 | Toxic! |
| Potassium Permanganate | Sigma-Aldrich | 223468 | For TLC staining. |
| Potassium phosphate dibasic | Sigma | 795496 | NA |
| Potassium phosphate monobasic | Sigma | 795488 | NA |
| Q Sepharose Fast Flow resin (GE Healthcare life sciences) | Fisher Scientific | 17-0515-01 | For anion exchange purification of enzyme |
| Sodium Chloride | Sigma | 71376 | NA |
| Tetrahydrofuran, anhydrous | Sigma-Aldrich | 186562 | NA |
| TMS-Diazomethane (2.0 M in hexanes) | Sigma-Aldrich | 362832 | Very toxic. |
| Tris-HCl | GoldBio | T-400 | NA |
| Also necessary: | |||
| Automatic flash purification system (we used a Buchi Reveleris X2) | Buchi | ||
| C18 HPLC column (Zorbax Eclipse XDB-C18) | Agilent | ||
| Centrifuge capable of 10,000 x g | |||
| Chiral HPLC Column (Lux cellulose-3), 250 x 4.6 mm, 5 µM, 1000 Å) | Phenomenex | ||
| General chemistry supplies: a 2 L separatory funnel, beakers and Erlenmeyer flasks with 1000-2000 L capacity, 20 mL vials, HPLC vials, small round-bottomed flasks and stir-bars. | |||
| HPLC (we use a Shimadzu Prominence LC-20AT analytical pump and SPD-20A UV-vis detector | Shimadzu | ||
| Nanodrop 2000 Spectrophotometer | Thermo-Fisher Scientific | ||
| NMR | NMR: Agilent DD2 spectrometer (500 MHz) | ||
| Rotary evaporator | Buchi | ||
| Sonic dismembrator or ultrasonic homogenizer | Cole-Parmer |
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