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Determination of Hydroquinone Dioxygenase Activity
对苯二酚双加氧酶活性的测定   

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Abstract

We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a hydroquinone dioxygenase, YaiA, described here, and a hydroquinone reductase, YaiB, described in a separate protocol. In Lactococcus lactis (L. lactis), these enzymes are encoded by the yahCD-yaiAB operon. The operon is induced by copper to prevent the synergistic toxicity of quinones and copper. The hydroquinone dioxygenase, YaiA, converts hydroquinones to 4-hydroxymuconic semialdehyde, using molecular oxygen as oxidant according to the reaction: hydroquinone + O2 → 4-hydroxymuconic semialdehyde + H+ We here describe two methods for measurements for hydroquinone dioxygenase activity, based on oxygen consumption measured with an oxygen electrode and the spectrophotometric detection of 4-hydroxymuconic semialdehyde. Both assays are conducted with crude cell extracts.

Materials and Reagents

  1. Hydroquinone dioxygenase from an Escherichia coli (E. coli) strain heterologously overexpressing such an enzyme from a plasmid (Mancini et al., 2015)
  2. Hydroquinone (100 mM in ethanol) (Sigma-Aldrich, catalog number: H9003 )
  3. 50 mM Tris-Cl [pH 7.5, 10% (v/v) glycerol]
  4. 20 mM Tris-Cl (pH 7.5)
  5. 10 mg/ml DNaseI (Sigma-Aldrich, catalog number: 11284932001 )
  6. 10 mM isopropyl-b-D-thiogalactoside (Sigma-Aldrich, catalog number: I6758 )
  7. 100 mM 4-hydroxybenzoate (Sigma-Aldrich, catalog number: H5501 )
  8. 100 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626 )
  9. 1 mM phenylmethylsulfonyl fluoride (Roche Diagnostics)
  10. Nitrocellulose filters (0.45 μm pore size)
  11. 100 mM hydroquinone (see Recipes)
  12. 50 mM Tris-Cl (see Recipes)
  13. 20 mM Tris-Cl (see Recipes)
  14. 10 mg/ml DNaseI (see Recipes)
  15. 10 mM isopropyl-β-D-thiogalactoside (see Recipes)
  16. 100 mM 4-hydroxybenzoate (see Recipes)
  17. 100 mM phenylmethylsulfonyl fluoride (see Recipes)

Equipment

  1. Thermostated spectrophotometer (Shimadzu, model: UV2600 or similar)
  2. Clark electrode (Warner Instruments, model: 1302 or any other commercial oxygraph)
  3. French Press

Procedure

  1. Preparation of crude E. coli extracts containing YaiA
    E. coli BL21(DE) containing plasmid pCA31 (Mancini et al., 2015) was grown aerobically in 300 ml of LB broth containing 100 μg/ml of ampicillin at 37 °C to an OD at 600 nm of 0.4. Then, 0.1 mM isopropyl-β-D-thiogalactoside was added, and growth was continued for 8 h at room temperature. Cells were harvested by centrifugation, and pellets suspended in 20 mM Tris-Cl (pH 7.5), containing 1 mM 4-hydroxybenzoate to stabilize protein as described elsewhere (Moonen et al., 2008), 1 mM phenylmethylsulfonyl fluoride, and 100 μg/ml of DNaseI. Cells were then broken by three passages through a French Press at 40 MPa, and cell debris was removed by centrifugation at 4 °C for 20 min at 90,000 x g. The supernatant was filtered through a 0.45 μm nitrocellulose filter and stored frozen at -20 °C until used.

  2. YaiA Activity assay by spectrophotometry
    1. Mix 50 mM Tris-Cl, pH 7.5, 10% (v/v) glycerol with 1 mg of crude extract in a total volume of 990 µl and warm to 30 °C in the spectrophotometer. This temperature was chosen since the origin of YaiA is L. lactis, which grows best at 30 °C.
    2. Zero the spectrophotometer and record the baseline spectrum from 300 to 400 nm. Start the reaction by adding 10 µl of 100 mM hydroquinone (final concentration 1 mM) and record spectra every 5 to 15 min, depending on the rate of the reaction. Calculate the specific activity from the increase in absorption at 320 nm, using an extinction coefficient for 4-hydroxymuconic semialdehyde of 11,000 M-1 cm-1 (Moonen et al., 2008; Spain and Gibson, 1991). Running spectra rather than measuring absorption at 320 nm is important to assured that the desired reaction is measured in the crude extracts. This should be further validated by calculating difference spectra (see Figure 1 and Note 1).

      Enzyme kinetics
      1. To determine the Km for hydroquinone, run enzyme reactions with 1, 3, 10, 30, 100, 300 and 1,000 µM hydroquinone.
      2. Determine the initial reaction rate, v0, for each reaction. If the reaction is linear, v0 is the slope of the linear regression of the data (e.g. calculated with the corresponding function built into Excel). If the reaction curve is sigmoidal, only the initial (highest) slope should be considered. Plot v0 versus the substrate concentrations, S.
      3.  Fit the curve to obtain the affinity for the substrate, Km, and the maximal velocity, vmax. Alternatively, plot v0 versus 1/S to obtain a Lineweaver-Burk plot. The regression line intercepts the ordinate at 1/vmax and the abscissa at -1/Km.

  3. YaiA activity assay by measuring oxygen consumption
    1. In a 1 ml chamber fitted with a Clark oxygen electrode or in any commercial oxygraph chamber, thermostated to 30 °C, mix 50 mM Tris-Cl (pH 7.5), 10% (v/v) glycerol, with 1 mg of crude extract.
    2. When a constant rate of oxygen consumption is attained (basal oxygen consumption due to other enzyme activities), inject 10 µl of 100 mM hydroquinone into the chamber and continue to record the oxygen consumption.
    3. Subtract the basal oxygen consumption from the rate observed with hydroquinone. Calculate the specific activity by calculating the oxygen consumption, based on the chamber volume and the oxygen content of the buffer, which can be taken from Estabrook (1967) or, if high precision is required calculated as described by Rasmussen and Rasmussen (2003). Under our conditions, buffer at 30 °C contains 222 µM O2.

      Enzyme kinetics
      1. To determine the Km for hydroquinone, run enzyme reactions with 1, 3, 10, 30, 100, 300 and 1,000 µM quinone.
      2. Determine the initial reaction rate, v0, for each reaction and plot v0 versus the substrate concentrations, S.
      3. Fit the curve to obtain the affinity for the substrate, Km, and the maximal velocity, vmax. Alternatively, plot 1/v0 versus 1/S to obtain a Lineweaver-Burk plot, from which Km and vmax can be determined.

Representative data



Figure 1. Absorption spectra of hydroquinone conversion to 4-hydroxymuconic semialdehyde. The lower curve was recorded at time 0 and the upper curve after 60 min of reaction. Inset, difference spectrum recorded between YaiA-containing extract incubated with and without 1 mM hydroquinone for 1 h, showing the absorption maximum of the reaction product at 320 nm. Axis labels are the same as in the main figure. Note that the spectra will look differently if substituted hydroquinones are used as the substrate for YaiA, but are not expected to change for different YaiA-like enzymes.


Figure 2. Oxygen consumption by hydroquinone dioxygenase activity in crude extracts. The reactions were induced by the addition of 1 mM hydroquinone at 2.5 min. Curves from top to bottom are: 1 mg of cytoplasmic extract from E. coli not expressing hydroquinone dioxygenase, extract heated to 95 °C for 5 min, and 1 mg of cytoplasmic extract from E. coli strain expressing YaiA of Lactococcus lactis from a plasmid.

Notes

  1. If difference spectra between unreacted and reacted extracts show the expected absorption spectrum for 4-hydroxymuconic semialdehyde, activity may be measured by continuous recording at 320 nm.
  2. Hydroquinone dioxygenase from L. lactis could not be purified in an active state and in crude cell lysates of L. lactis, the activity was too low to be detected.
  3. 4-hydroxymuconic semialdehyde is very labile and difficult to isolate and characterize. However, no change in absorbance was observed over at least one hour once the reaction had come to completion.
  4. Hydroquinone must be prepared fresh on the day of use due to the spontaneous auto-oxidation to benzoquinone in the presence of oxygen. No significant autoxidation of the stock solution was observed within 8 h.

Recipes

  1. 100 mM hydroquinone
    Dissolved in 100% ethanol
    Stored at room temperature in the dark
  2. 50 mM Tris-Cl (pH 7.5)
    10% (v/v) glycerol
    Stored at 4 °C or autoclave to prevent bacterial growth
  3. 20 mM Tris-Cl (pH 7.5)
    Stable at room temperature
  4. 10 mg/ml DNaseI
    Stable at -20 °C
  5. 10 mM isopropyl-β-D-thiogalactoside
    Stable at -20 °C
  6. 100 mM 4-hydroxybenzoate
    Stable at room temperature
  7. 100 mM phenylmethylsulfonyl fluoride
    Dissolve in 100% dimethylsulfoxide and store at -20 °C

Acknowledgments

This work was supported by Russian Federation Government Grant 14.Z50.31.0011 to leading scientists. The procedure has previously been described in Mancini et al. (2015).

References

  1. Estabrook, R. W. (1967). Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol 10, 41-47.
  2. Mancini, S., Abicht, H. K., Gonskikh, Y. and Solioz, M. (2015). A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403. Mol Microbiol 95(4): 645-659.
  3. Moonen, M. J., Kamerbeek, N. M., Westphal, A. H., Boeren, S. A., Janssen, D. B., Fraaije, M. W. and van Berkel, W. J. (2008). Elucidation of the 4-hydroxyacetophenone catabolic pathway in Pseudomonas fluorescens ACB. J Bacteriol 190(15): 5190-5198.
  4. Rasmussen, H. N. and Rasmussen, U. F. (2003). Oxygen solubilities of media used in electrochemical respiration measurements. Anal Biochem 319(1): 105-113.
  5. Spain, J. C. and Gibson, D. T. (1991). Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp. Appl Environ Microbiol 57(3): 812-819.

简介

我们最近展示了存在于Firmicutes中的醌解毒途径。 它基于两种酶活性,即本文所述的氢醌双加氧酶,YaiA和在单独方案中描述的氢醌还原酶,YaiB。 在乳酸乳球菌(乳酸乳杆菌)中,这些酶由yahCD-yaiAB 操纵子编码。 操纵子由铜诱导以防止醌和铜的协同毒性。 根据以下反应,氢醌双加氧酶YaiA使用分子氧作为氧化剂将氢醌转化为4-羟基粘康酸半醛:氢醌+ O 2→4-羟基粘康酸半醛+ H + 我们在这里描述了用于测量氢醌双加氧酶活性的两种方法,基于用氧电极测量的氧消耗和4-羟基粘康酸半醛的分光光度检测。 两种测定用粗细胞提取物进行。

材料和试剂

  1. 来自大肠杆菌的大肠杆菌双加氧酶(大肠杆菌)菌株从质粒异源过表达这种酶(Mancini等人,2015) br />
  2. 氢醌(100mM,在乙醇中)(Sigma-Aldrich,目录号:H9003)
  3. 50mM Tris-Cl [pH7.5,10%(v/v)甘油]
  4. 20mM Tris-Cl(pH7.5)
  5. 10mg/ml DNaseI(Sigma-Aldrich,目录号:11284932001)
  6. 10mM异丙基-β-D-硫代半乳糖苷(Sigma-Aldrich,目录号:I6758)
  7. 100mM 4-羟基苯甲酸酯(Sigma-Aldrich,目录号:H5501)
  8. 100mM苯甲基磺酰氟(Sigma-Aldrich,目录号:P7626)
  9. 1mM苯甲基磺酰氟(Roche Diagnostics)
  10. 硝化纤维素过滤器(0.45μm孔径)
  11. 100 mM氢醌(参见配方)
  12. 50 mM Tris-Cl(参见配方)
  13. 20 mM Tris-Cl(参见配方)
  14. 10 mg/ml DNaseI(见配方)
  15. 10mM异丙基-β-D-硫代半乳糖苷(参见配方)
  16. 100 mM 4-羟基苯甲酸(参见配方)
  17. 100mM苯甲基磺酰氟(参见配方)

设备

  1. 恒温分光光度计(Shimadzu,型号:UV2600或类似)
  2. Clark电极(Warner Instruments,型号:1302或任何其他商业氧测量仪)
  3. 法语出版社

程序

  1. 制备粗E。 含有YaiA的大肠杆菌提取物
    E。将含有质粒pCA31(Mancini等人,2015)的大肠杆菌BL21(DE)在37℃下在含有100μg/ml氨苄青霉素的300ml LB肉汤中有氧生长至OD在600nm为0.4。然后,加入0.1mM异丙基-β-D-硫代半乳糖苷,并在室温下继续生长8小时。通过离心收获细胞,并将小球悬浮于含有1mM 4-羟基苯甲酸酯以稳定蛋白质的20mM Tris-Cl(pH7.5)中,如别处所述(Moonen等人,2008),1mM苯甲基磺酰氟和100μg/ml DNaseI。然后通过弗氏压碎器(French Press)在40MPa下破碎细胞,通过在4℃下以90,000×g离心20分钟除去细胞碎片。将上清液通过0.45μm硝化纤维素过滤器过滤,并在-20℃冷冻储存直至使用。

  2. YaiA通过分光光度法的活性测定
    1. 将50mM Tris-Cl,pH 7.5,10%(v/v)甘油与1mg粗提取物混合  在总体积为990μl并在分光光度计中温热至30℃。  选择该温度是因为YaiA的来源是L。乳酸, 其在30℃下最好生长
    2. 零分光光度计和记录 基线光谱从300到400 nm。通过添加开始反应 10μl100mM氢醌(终浓度1mM)并记录 光谱每5至15分钟,取决于反应的速率。 在320处从吸收的增加计算比活性 使用4-羟基粘康酸半醛的消光系数  (Moonen等人,2008; Spain和Gibson,1991)。运行  光谱而不是测量320nm处的吸收是重要的 确保在粗提取物中测量所需的反应。 这应该通过计算差异光谱进一步验证 图1和注1)。

      酶动力学
      1. 为了测定氢醌的K m,使用1,3,10,30,100,300和1,000μM氢醌进行酶反应。
      2. 确定每个反应的初始反应速率, v 0 。如果 反应是线性的, v 0 是数据的线性回归的斜率  (例如,使用Excel中内置的相应函数计算)。如果 反应曲线是S形的,只有初始(最高)斜率应该 被考虑。 底物浓度S.
      3.  拟合曲线以获得对底物的亲和力,K m 最大速度, v max 。 或者,绘制 v 0 与 1/S, Lineweaver-Burk情节。 回归线截取纵坐标 1/em> v max ,横坐标在-1/K

  3. 通过测量氧消耗的YaiA活性测定
    1. 在装有Clark氧电极的1ml室中或任何其中 商业PCR仪,恒温至30℃,混合50mM Tris-Cl (pH7.5),10%(v/v)甘油,1mg粗提取物
    2. 当一个 达到恒定的耗氧量(基础氧 由于其他酶活性消耗),注射10μl的100mM 氢醌进入腔室并继续记录氧气 消耗
    3. 从速率中减去基础氧消耗 用氢醌观察。 通过计算比活性 基于所述室容积计算所述氧消耗 缓冲液的氧含量,其可以取自Estabrook(1967) 或者,如果需要计算高精度,如Rasmussen所述 和Rasmussen(2003)。 在我们的条件下,30℃下的缓冲液含有222   μMO 2

      酶动力学
      1. 为了确定氢醌的K m,使用1,3,10,30,100,300和1,000μM的醌进行酶反应。
      2. 确定每个反应的初始反应速率 v 0 ,并且绘制 v 0 em>底物浓度S.
      3. 拟合曲线以获得对底物的亲和力K m, 最大速度, v max 。 或者,绘图1/ 0 与 1/S Lineweaver-Burk图,从中可以确定K sub和v em。 max

代表数据



图1. 氢醌转化为4-羟基粘康酸半醛的吸收光谱在时间0记录下方曲线,在反应60分钟后记录下方曲线。插图,在含有和不含1mM氢醌温育1小时的含有YaiA的提取物之间记录的差谱,显示在320nm处反应产物的最大吸收。轴标签与主图相同。注意,如果取代的氢醌用作YaiA的底物,则光谱将看起来不同,但是预期不会因不同的YaiA样酶而改变。

图2。粗提取物中氢醌双加氧酶活性的氧消耗。通过在2.5分钟时加入1mM氢醌诱导反应。曲线从顶部到底部是:1mg来自E的细胞质提取物。不表达氢醌双加氧酶的大肠杆菌,加热至95℃的提取物5分钟,和1mg来自E的细胞质提取物。表达来自质粒的乳酸乳球菌的YaiA的大肠杆菌菌株。

笔记

  1. 如果未反应和反应的提取物之间的差异光谱显示4-羟基粘康酸半醛的预期吸收光谱,则活性可以通过在320nm下连续记录来测量。
  2. 来自乳酸乳球菌的氢醌双加氧酶不能以活性状态和在L的粗细胞裂解物中纯化。 乳酸,活性太低而无法检测。
  3. 4-羟基粘康酸半醛非常不稳定并且难以分离和表征。 然而,一旦反应完成,在至少一小时内没有观察到吸光度的变化
  4. 氢醌必须在使用当天新鲜制备,因为在氧存在下自发自发氧化为苯醌。 在8小时内没有观察到储备溶液的显着自氧化

食谱

  1. 100mM氢醌 溶于100%乙醇
    在室温下贮存在黑暗中
  2. 50mM Tris-Cl(pH7.5) 10%(v/v)甘油 储存在4℃或高压灭菌以防止细菌生长
  3. 20mM Tris-Cl(pH7.5) 在室温下稳定
  4. 10 mg/ml DNaseI
    在-20°C稳定
  5. 10mM异丙基-β-D-硫代半乳糖苷 在-20°C稳定
  6. 100mM 4-羟基苯甲酸酯 在室温下稳定
  7. 100mM苯甲基磺酰氟 溶于100%二甲基亚砜中,贮存于-20℃

致谢

这项工作是由俄罗斯联邦政府赠款14.Z50.31.0011支持领先的科学家。 该程序先前已在Mancini等人(2015)中描述。

参考文献

  1. Estabrook,R.W。(1967)。线粒体呼吸控制和ADP:​​O比的极谱测量。 Methods Enzymol 10,41-47。
  2. Mancini,S.,Abicht,H.K.,Gonskikh,Y。和Solioz,M。(2015)。 铜诱导的醌降解途径提供针对乳酸乳球菌中铜/醌综合应激的保护 IL1403。 Mol Microbiol 95(4):645-659。
  3. Moonen,M.J.,Kamerbeek,N.M.,Westphal,A.H.,Boeren,S.A.,Janssen,D.B.,Fraaije,M.W.and van Berkel,W.J。(2008)。 在荧光假单胞菌 ACB中阐明4-羟基苯乙酮分解代谢途径。/a> J Bacteriol 190(15):5190-5198。
  4. Rasmussen,H.N.和Rasmussen,U.F。(2003)。 用于电化学呼吸测量的培养基的氧溶解度。 em> 319(1):105-113。
  5. Spain,J.C。和Gibson,D.T。(1991)。 莫拉氏菌中的对硝基苯酚的生物降解途径 Appl Environ Microbiol 57(3):812-819。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mancini, S. and Solioz, M. (2015). Determination of Hydroquinone Dioxygenase Activity. Bio-protocol 5(17): e1580. DOI: 10.21769/BioProtoc.1580.
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