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Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, also Marfey’s reagent) for the characterization of novel amino acid racemases. The protocol described here was designed to obtain accurate kinetic parameters (kcat, KM values). Enzyme concentrations and reaction times were optimized so as to minimize the reverse reaction, which can confound results when measuring racemase reactions.

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Amino Acid Racemase Enzyme Assays
氨基酸消旋酶分析

微生物学 > 微生物生物化学 > 蛋白质 > 活性
作者: Atanas D. Radkov
Atanas D. RadkovAffiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, USA
Bio-protocol author page: a1313
 and Luke A. Moe
Luke A. MoeAffiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, USA
For correspondence: luke.moe@uky.edu
Bio-protocol author page: a1314
Vol 4, Iss 9, 5/5/2014, 2906 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1112

[Abstract] Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, also Marfey’s reagent) for the characterization of novel amino acid racemases. The protocol described here was designed to obtain accurate kinetic parameters (kcat, KM values). Enzyme concentrations and reaction times were optimized so as to minimize the reverse reaction, which can confound results when measuring racemase reactions.

[Abstract]

Materials and Reagents

  1. Escherichia coli (E. coli) Rosetta 2 (DE3) cell line
  2. pET overexpression system
  3. His-tag and Ni2+-NTA affinity chromatography (HIS-Select Nickel Affinity Gel) (Sigma-Aldrich, catalog number: P6611 )
  4. AA substrates
    Note: Each enantiomer of the 19 chiral proteinogenic AAs, and the four epimers of hydroxyproline, are prepared in 50 mM HEPES buffer (pH 7.4) with any co-factors (PLP at 20 μM was used in our case.).
  5. 0.5% solution (w/v, in acetone) of Marfey’s reagent (Sigma-Aldrich, catalog number: 71478 )
  6. 1 M NaHCO3
  7. 2 M HCl
  8. 2 M NaOH
  9. HPLC buffer (0.05 M TEAP buffer, pH 3.0) (see Recipes)

Equipment

  1. Bottle top filter (polystyrene) (Corning, catalog number: 430513 ) (filter used in TEAP buffer preparation)
  2. Syringe filter (PTFE) (Tisch Scientific, catalog number: SF14466 ) (filter used in HPLC sample preparation)
  3. Heat block (set at 37 °C for enzyme reactions)
  4. Heat block (set at 40 °C for derivatization of reaction products)
  5. 2 ml microfuge tubes (for the reaction, derivatization, and dilution of derivatized products)
  6. Amber HPLC vials with caps, syringes (1 ml), needles, and filters (0.22 μm polytetrafluoroethylene, PTFE)
  7. Waters Nova-Pak (C18 column) (3.9 mm by 150 mm)

Software

  1. Microsoft Excel

Procedure

  1. Enzyme preparation and storage
    1. Enzyme was overexpressed using E. coli Rosetta 2 (DE3) cell line and the pET overexpression system, His-tag and Ni2+-NTA affinity chromatography [for specifics, please see Goodlett et al. (1995)].
    2. After chromatography and buffer exchange, the enzyme was divided into 50 μl aliquots and was snap-frozen before long-term storage at -80 °C.

  2. Enzyme assays
    1. Enzyme reactions were performed in 2 ml microfuge tubes.
    2. 199 μl of AA solution in 50 mM HEPES (pH 7.4) plus cofactors (if needed) were placed in each reaction tube.
      1. 20 μM pyridoxal-5’-phosphate (PLP) was added based on bioinformatic evidence that the enzyme has a PLP-binding motif.
    3. To pre-condition the assay solution, the assay tubes were kept at 37 °C for 5 min before enzyme addition.
    4. Assay was initiated by the addition of the purified enzyme to a final volume of 200 μl.
      1. The final enzyme concentration was determined empirically and varied based on the particular substrate used. In our case, between 1 μM and 2 μM final enzyme concentrations were used to determine relative substrate specificity, and between 1 nM and 2 μM - to calculate the enzyme kinetics. For further specifics, please see Goodlett et al. (1995).
      2. The molar concentration of enzyme used was kept at 10% or less of the initial substrate concentration.
    5. Assay was performed at 37 °C for 1 min.
      1. The assay duration was chosen so that only 10% or less of the initial substrate was consumed in order to minimize the occurrence of the reverse reaction.
    6. To quench the assay, 40 μl of 2 M HCl was added and mixed by pipetting.
    7. In preparation for derivatization, 40 μl 2 M NaOH was added to neutralize the acid.
    8. 50 μl of the neutralized, quenched assay was transferred to a new 2 ml microfuge tube.
    9. 100 μl of 0.5% Marfey’s reagent in acetone was added.
    10. 20 μl of 1 M NaHCO3 was added to make the solution alkaline, and the contents were mixed via pipetting.
    11. Derivatization was performed on a heat block at 40 °C for 1 h.
      1. During this time, fresh 1 L HPLC buffer was prepared and passed through 0.22 μm bottle top filter every time before sample analysis.
      2. Also, 900 μl of 80% 0.05 M TEAP buffer (pH 3.0) and 20% acetonitrile (HPLC grade) was placed in new 2 ml microfuge tubes in preparation for the 10-fold dilution of the derivatized products.
    12. Derivatization reaction was briefly centrifuged to collect any condensation from the lid of the microfuge tube.
    13. Reaction was cooled at room temperature for 5 min.
    14. 100 μl of the reaction was mixed with the solution in step B11b to prepare a 10-fold dilution.
    15. The diluted derivatization reaction was passed through a PTFE 0.22 μm syringe filter and placed into an amber HPLC vial.
      1. Derivatization reactions should be kept in an amber vial to prevent photochemical decomposition of the absorbing chromophore (Marfey, 1984). In our case, assays were analyzed within 48 h after derivatization.
    16. The same procedure as above was followed (the enzyme addition was omitted.) to determine AA elution times and prepare standard curves.

  3. HPLC analysis
    1. The specific column employed in our case was Waters Nova-Pak, C18 column.
    2. 10 μl of the diluted derivatized reaction (from step B15) was analyzed.
    3. Flow rate was 0.5 ml/min.
    4. Derivatized products were detected at 340 nm.
    5. Several gradients were employed for the separation of different enantiomeric pairs.
      1. For a description of the gradients, please refer to the methods section and supplementary material of the original publication by Radkov and Moe (2013).

  4. Data analysis
    1. Due to differences in the derivatization efficiency between enantiomeric pairs, derivatized D-AAs yield a higher UV response relative to L-AAs. The data provided by Goodlett et al. was used to adjust for these differences before preparing standard curves and calculating specific activities.
      1. Example chromatogram



    2. The Solver function in Microsoft Excel was used to perform non-linear curve fitting to the Michaelis-Menten equation.
      1. An example dataset (attached here) is provided as a supplementary file.

Recipes

  1. HPLC buffer (0.05 M TEAP buffer, pH 3.0)
    Solution of 0.05 M triethylamine was prepared and the pH was adjusted using concentrated phosphoric acid.

Acknowledgments

This protocol was adapted from Radkov and Moe, (2013). This work was supported in part by grant 2011-67020-30195 from the USDA National Institute of Food and Agriculture.

References

  1. Goodlett, D. R., Abuaf, P. A., Savage, P. A., Kowalski, K. A., Mukherjee, T. K., Tolan, J. W., Corkum, N., Goldstein, G. and Crowther, J. B. (1995). Peptide chiral purity determination: hydrolysis in deuterated acid, derivatization with Marfey's reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry. J Chromatogr A 707(2): 233-244.
  2. Marfey, P. (1984). Determination ofD-amino acids. II. Use of a bifunctional reagent, 1, 5-difluoro-2, 4-dinitrobenzene. Carlsberg Res Communi 49(6): 591-596.
  3. Radkov, A. D. and Moe, L. A. (2013). Amino acid racemization in Pseudomonas putida KT2440. J Bacteriol 195(22): 5016-5024.

材料和试剂

  1. 大肠杆菌(大肠杆菌)Rosetta 2(DE3)细胞系
  2. pET过表达系统
  3. His标签和Ni 2+ 2+ -NTA亲和层析(HIS-Select Nickel Affinity Gel)(Sigma-Aldrich,目录号:P6611)。
  4. AA底物
    注意:在具有任何辅因子的50mM HEPES缓冲液(pH 7.4)中制备19种手性蛋白质AAs的每种对映异构体和羟脯氨酸的四种差向异构体(在我们的情况下使用20μM的PLP)。
  5. 的Marfey试剂(Sigma-Aldrich,目录号:71478)的0.5%溶液(w/v,在丙酮中)
  6. 1 M NaHCO 3 3/
  7. 2 M HCl
  8. 2 M NaOH
  9. HPLC缓冲液(0.05M TEAP缓冲液,pH 3.0)(参见Recipes)

设备

  1. 瓶顶过滤器(聚苯乙烯)(Corning,目录号:430513)(用于TEAP缓冲液制备的过滤器)
  2. 注射器过滤器(PTFE)(Tisch Scientific,目录号:SF14466)(用于HPLC样品制备的过滤器)
  3. 热块(设定在37℃下酶反应)
  4. 加热块(设定在40℃,用于衍生反应产物)
  5. 2ml微量离心管(用于衍生化产物的反应,衍生化和稀释)
  6. 带有盖,注射器(1ml),针和过滤器(0.22μm聚四氟乙烯,PTFE)的琥珀色HPLC小瓶
  7. Waters Nova-Pak(C18 C18柱)(3.9mm×150mm)

软件

  1. Microsoft Excel

程序

  1. 酶制剂和储存
    1. 使用E过表达酶。 大肠杆菌Rosetta 2(DE3)细胞系和pET过表达系统,His标签和Ni 2+ 2 + -NTA亲和层析[具体参见Goodlett 。 (1995)]。
    2. 在色谱和缓冲液交换后,将酶分成50μl等分试样,在-80℃长期储存之前快速冷冻。

  2. 酶测定
    1. 酶反应在2ml微量离心管中进行
    2. 将199μl在50mM HEPES(pH 7.4)中的AA溶液加上辅因子(如果需要)置于每个反应管中。
      1. 基于生物信息学证据,加入20μM吡哆醛-5'-磷酸(PLP),该酶具有PLP结合基序。
    3. 为了预处理测定溶液,将测定管在37℃下保持5分钟,然后加入酶
    4. 通过加入纯化的酶至终体积为200μl开始测定。
      1. 最终酶浓度根据经验确定并且基于所使用的特定底物而变化。 在我们的情况下,使用1μM和2μM之间的最终酶浓度来确定相对底物特异性,并且在1nM和2μM之间以计算酶动力学。 有关详细信息,请参阅Goodlett 等。 (1995)。
      2. 所用酶的摩尔浓度保持在初始底物浓度的10%或更低
    5. 测定在37℃进行1分钟。
      1. 选择测定持续时间使得仅消耗10%或更少的初始底物以使反向反应的发生最小化。
    6. 为了淬灭测定,加入40μl2M HCl并通过吸移混合
    7. 在准备衍生化时,加入40μl2M NaOH以中和酸
    8. 将50μl中和的淬灭测定转移到新的2ml微量离心管中
    9. 加入100μl0.5%Marfey试剂的丙酮溶液
    10. 加入20μl1M NaHCO 3以使溶液呈碱性,并通过移液混合内容物。
    11. 在加热块上在40℃下进行衍生化1小时。
      1. 在此期间,制备新鲜的1L HPLC缓冲液,并在样品分析前每次通过0.22μm瓶顶过滤器
      2. 此外,将900μl80%0.05M TEAP缓冲液(pH 3.0)和20%乙腈(HPLC级)置于新的2ml微量离心管中,以制备衍生化产物的10倍稀释液。
    12. 将衍生化反应物简单离心以收集微量离心管的盖子上的任何冷凝物
    13. 将反应物在室温下冷却5分钟
    14. 将100μl反应物与步骤B11b中的溶液混合以制备10倍稀释液
    15. 使稀释的衍生化反应通过PTFE0.22μm注射器过滤器,并放入琥珀色HPLC小瓶中。
      1. 衍生化反应应保持在琥珀色小瓶中以防止吸收性发色团的光化学分解(Marfey,1984)。 在我们的情况下,在衍生化后48小时内分析测定。
    16. 进行与上述相同的操作(省略酶添加),以确定AA洗脱时间并制备标准曲线。

  3. HPLC分析
    1. 在我们的情况中使用的特定柱是Waters Nova-Pak,C 18柱
    2. 分析10μl稀释的衍生化反应(来自步骤B15)
    3. 流速为0.5ml/min
    4. 在340nm检测衍生化产物
    5. 使用几种梯度来分离不同的对映异构体对。
      1. 有关渐变的描述,请参阅方法部分 和Radkov和Moe的原始出版物的补充材料   (2013年)。

  4. 数据分析
    1. 由于对映异构体对之间的衍生化效率的差异,衍生的D-AAs相对于L-AAs产生更高的UV响应。 由Goodlett等人提供的数据用于在准备标准曲线和计算比活度之前调整这些差异。
      1. 色谱图示例



    2. Microsoft Excel中的求解器函数用于对Michaelis-Menten方程执行非线性曲线拟合。
      1. 示例数据集 (附加 < a>)作为补充文件提供。

食谱

  1. HPLC缓冲液(0.05M TEAP缓冲液,pH 3.0) 制备0.05M三乙胺的溶液,并使用浓磷酸调节pH

致谢

该协议改编自Radkov和Moe,(2013)。 这项工作部分得到美国农业部国家粮食和农业研究所的授权2011-67020-30195的支持。

参考文献

  1. Goodlett,D.R.,Abuaf,P.A.,Savage,P.A.,Kowalski,K.A.,Mukherjee,T.K.,Tolan,J.W.,Corkum,N.,Goldstein,G.and Crowther,J.B。(1995)。 肽手性纯度测定:在氘代酸中水解,用Marfey试剂衍生化,使用高效液体分析色谱 - 电喷雾电离 - 质谱法。 J Chromatogr A 707(2):233-244。
  2. Marfey,P。(1984)。 D-氨基酸的测定。 II。使用双功能试剂,1,5-二氟-2,4-二硝基苯。 Carlsberg Res  Communi 49(6):591-596。
  3. Radkov,A. D.和Moe,L.A。(2013)。 恶臭假单胞菌(Pseudomonas putida) KT2440中的氨基酸外消旋化。 J Bacteriol 195(22):5016-5024。
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How to cite this protocol: Radkov, A. D. and Moe, L. A. (2014). Amino Acid Racemase Enzyme Assays. Bio-protocol 4(9): e1112. DOI: 10.21769/BioProtoc.1112; Full Text



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