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Assay for GTP Cyclohydrolase II Activity in Bacterial Extracts
细菌提取物中GTP环化水解酶II活性分析   

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Abstract

Riboflavin is the precursor of flavin nucleotides FMN and FAD, they play significant roles in all organisms. GTP is the initial precursor on riboflavin biosynthesis pathway and GTP cyclohydrolase II catalyzes the first step of this pathway. It converts GTP to 2,5-diamino-6-ribosylamino-4 (3H) -pyrimidinone 5'-phosphate. This protocol provides a reliable and fast method to assay GTP cyclohydrolase II activity from crude bacterial extracts. The product of the reaction catalyzed by GTP cyclohydrolase II, 2,5-diamino-6-ribosylamino-4 (3H) -pyrimidinone 5'-phosphate, is converted to its fluorescent derivative 6,7-dimethylpterin, which is then separated on a XTerra MS C18 column and detected using fluorescence HPLC system.

Materials and Reagents

  1. Sinorhizobium meliloti (S. meliloti 1021, Galibert et al., 2001)
    Note: The protocol can be also applied to other bacteria.
  2. Bio-Rad Protein Assay Kit I (Sigma-Aldrich, catalog number: 500-0001 )
  3. 50 mM Tris-HCl buffer (pH 7.5) (5 ml per sample)
  4. 1x BugBuster reagent (Novagen, catalog number: 70921 )
  5. 0.5 M EDTA (pH 8.0) (2.5 µl per sample)
  6. GTP (Guanosine 5’-triphosphate sodium salt hydrate) (Sigma-Aldrich, catalog number: G8877 )
  7. HPLC Standard: 6,7-Dimethylpterin (Schircks Laboratories, catalog number: 11.503 )
  8. Biotin
  9. YMB medium (Somerville and Kahn, 1983) (see Recipes) (1 plate per sample)
  10. MMNH4 medium (Somerville and Kahn, 1983) (see Recipes) (13 ml per sample)
  11. Lysis buffer (see Recipes) (1 ml per sample)
  12. Desalting buffer (see Recipes) (3 ml per sample)
  13. RibA assay buffer (see Recipes) (2.5 µl per sample)
  14. Derivatization reagent (see Recipes) (50 µl per sample)
  15. HPLC mobile phase (see Recipes)
    Note: Except as otherwise noted, all other chemicals were obtained from Sigma-Aldrich.

Equipment

  1. 14 ml Falcon tubes (BD Biosciences, catalog number: 352059 )
  2. Eppendorf 1.7 ml tubes (Thermo Fisher Scientific, catalog number: 14-222-168 )
  3. 0.22 µm syringe filter (Microsolv Technology, catalog number: 58022-N04-C )
  4. 2 ml Zeba Spin Desalting Columns (Thermo Fisher Scientific, catalog number: 87768 )
  5. Mini-centrifuge
  6. Centrifuge
  7. Shaker
  8. HPLC: Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector
  9. XTerra MS C18 column (4.6 x 100 mm, 5 µm) (Waters, part number: 186000486 )

Procedure

  1. Cell extract preparation
    1. Grow S. meliloti on YMB plate for 48 h at 30 °C. The optimal temperature for S. meliloti growth is between 28 °C and 30 °C.
    2. Inoculate S. meliloti from the stock YMB plate to an OD600 of ~0.1 in 3 ml MMNH4 medium.
    3. Grow cells for 48 h at 30 °C, 250 rpm in 14 ml Falcon or glass tubes.
    4. Dilute the cells 20-fold into 10 ml fresh MMNH4 medium and grow overnight at 30 °C, 250 rpm in 50 ml glass tubes or flasks.  
    5. Harvest cells from 10 ml culture by centrifugation at 3,600 x g for 15-20 min at 4 °C in 14 ml Falcon tubes.  
    6. Discard the supernatant and wash the cells with 5 ml 50 mM Tris buffer at 4 °C.
    7. Resuspend the pellet in 1 ml of lysis buffer at 4 °C.
    8. Incubate at room temperature for 15 min, without shaking.
    9. Centrifuge the lysate at maximum speed for 15 min at 4 °C.
    10. Measure protein concentration in the cell lysates using Bio-Rad Protein Assay Kit.

  2. Enzymatic assay
    This assay protocol was adapted from a method for assaying GTP cyclohydrolase II activity in the purified enzyme (Bacher et al., 1997) and modified for using with bacterial extracts.
    1. Desalt the cell lysate using 2 ml Zeba Spin Desalting Columns following the manufacturer’s instructions. The column was equilibrated with 3 ml desalting buffer.
    2. Add 25 µl of the desalted whole cell lysate form the previous step in a total volume of 50 µl reaction mixture containing 10 µl 5 x RibA assay buffer, 5 µl 10 mM GTP (prepare GTP in ultrapure water).
    3. Incubate the assay at 37 °C for 30 min.
    4. Terminate the assay by adding 2.5 µl EDTA (0.5 M, pH 8.0).
    5. Derivatize the product by adding 50 µl of the derivatization reagent, followed by incubation at 70 °C for 20 min.
    6. Clear the samples by centrifugation at 3,000 x g for 10 min at 4 °C.
    7. Filter the supernatant using a 0.22 µm syringe filter.
    8. Analyze the derivatization products using HPLC with fluorescence detection.

  3. HPLC purification and signal detection
    1. Separate the fluorescent products on a XTerra MS C18 column (4.6 x 100 mm, 5 µm) using a Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector. Excitation and emission wavelengths are 330 nm and 435 nm, respectively.

  4. Data analysis
    1. Quantify the products by comparison to standards. A standard curve was obtained by running sequential dilution of 6,7-dimethylpterin (0.25 nM, 2.5 nM, 25 nM, 0.25 µm, 2.5 µM and 25 µM) on HPLC.
    2. Normalize the enzyme activity against protein concentration and assay incubation time in nmol/min/mg protein.
    3. Perform three replicates on each assay. Data is the average ± S.E. of three replicates.
      Note: The activities of GTP cyclohydrolase II vary in different organisms. The activity of purified recombinant enzymes have been reported from about 0.1- 182 nmol/min/mg protein, the activity in bacterial extracts can be lower (Herz et al., 2000; Kaiser et al., 2002; Yurgel et al., 2014).

Representative data





Figure 1. HPLC chromatograms of 2.5 µM 6,7-dimethylpterin standard (A), product of GTP cyclohydrolase II assay from bacterial extracts (B) and co-elution of product from GTP cyclohydrolase II assay with 6,7-dimethylpterin standard (C).
HPLC mobile phase: 10% methanol and 90% phosphoric acid (v/v). Excitation and emission wavelengths were 330 nm and 435 nm respectively.

Recipes

  1. YMB media for Rhizobium

    1 L
    Concentration
    Yeast Extract
    1 g

    Mannitol
    10 g
    54.9 mM
    Agar
    15 g


    Autoclave, cool to 55 °C, then add
    YMB Salt I
    10 ml
    YMB Salt II
    10 ml

    YMB Salt I
    1 L
    Concentration
    K2HPO4
    50 g
    287.06 mM
    NaCl
    10 g
    171.15 mM
    d-H2O
    960 ml


    YMB Salt II
    1 L
    Concentration
    MgSO4.7H2O
    20 g
    81.11 mM
    d-H2O
    1 L


  2. MMNH4 (minimal mannitol ammonia media for Rhizobium)

    per 1 L
    Concentration
    Mannitol
    10.0 g
    54.9 mM
    NH4Cl
    0.5 g
    9.34 mM
    Agar (for plates preparation)
    15.0 g

    d-H2O
    970 ml


    Autoclave, cool to 55 °C, then add:
    Biotin (0.2 mg/ml in 50% EtOH)
    1.0 ml
    Thiamine (2 mg/ml), filter sterilized
    1.0 ml
    Min Man Salts I
    10.0 ml
    Min Man Salts II
    10.0 ml

    Min Man Salts I

    per 1 L
    Concentration
    K2HPO4
    100 g
    574.12 mM
    KH2PO4
    100 g
    734.8 mM
    Na2SO4
    25 g
    174.8 mM
    d-H2O
    1 L

    Min Man Salt II

    per 1 L
    Concentration
    FeCl3.6H2O
    1.0 g
    3.7 mM
    Concentrated HCl
    adjust pH to ~7.0 (~1drop)
    CaCl2.2H2O
    10.0 g
    68 mM
    MgCl2.6H2O
    25.0 g
    123 mM
    d-H2O
    1 L
    Autoclave

  3. Lysis buffer
    50 mM Tris-HCl (pH 7.5)
    10 mM MgCl2
    1 mM Tris (hydroxypropyl) phosphine
    1x BugBuster reagent
  4. Desalting buffer
    50 mM Tris-HCl (pH 7.5)
    1 mM Tris (hydroxypropyl) phosphine
    10% glycerol
  5. RibA assay buffer
    100 mM Tris-HCl (pH 8.5) buffer
    5 mM MgCl2
    5 mM dithiothreitol (DTT)
  6. Derivatization reagent
    1% (v/v) diacetyl
    15% (w/v) trichloroacetic acid
  7. HPLC mobile phase
    10% (v/v) methanol
    90% phosphoric acid (27 mM phosphoric acid)

Acknowledgments

This protocol was adapted from Yurgel et al., 2014. This work was supported by the Agricultural Research Center (WNP-00773) at Washington State University and grant DE-FG0396ER20225 Vol. 27, No. 5, 2014 / 445 from the Energy Biosciences Program at the United States Department of Energy, and by grant NSF-MCB 1052492 to S. Rajamani. This activity was funded, in part, with an Emerging Research Issues Internal Competitive grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences and Biologically-Intensive Agriculture and Organic Farming (BIOAg) Internal Competitive grant from The Center for Sustaining Agriculture and Natural Resources (CSANR) at Washington State University to S. Yurgel. We thank M. Kahn for discussions and the Washington State University Laboratory for Biotechnology and Bioanalysis for sequencing support.

References

  1. Bacher, A., Richter, G., Ritz, H., Eberhardt, S., Fischer, M. and Krieger, C. (1997). Biosynthesis of riboflavin: GTP cyclohydrolase II, deaminase, and reductase. Methods Enzymol 280: 382-389.
  2. Galibert, F., Finan, T. M., Long, S. R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M. J., Becker, A., Boistard, P., Bothe, G., Boutry, M., Bowser, L., Buhrmester, J., Cadieu, E., Capela, D., Chain, P., Cowie, A., Davis, R. W., Dreano, S., Federspiel, N. A., Fisher, R. F., Gloux, S., Godrie, T., Goffeau, A., Golding, B., Gouzy, J., Gurjal, M., Hernandez-Lucas, I., Hong, A., Huizar, L., Hyman, R. W., Jones, T., Kahn, D., Kahn, M. L., Kalman, S., Keating, D. H., Kiss, E., Komp, C., Lelaure, V., Masuy, D., Palm, C., Peck, M. C., Pohl, T. M., Portetelle, D., Purnelle, B., Ramsperger, U., Surzycki, R., Thebault, P., Vandenbol, M., Vorholter, F. J., Weidner, S., Wells, D. H., Wong, K., Yeh, K. C. and Batut, J. (2001). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293(5530): 668-672.
  3. Herz, S., Eberhardt, S. and Bacher, A. (2000). Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase. Phytochemistry 53(7): 723-731.
  4. Kaiser, J., Schramek, N., Eberhardt, S., Puttmer, S., Schuster, M. and Bacher, A. (2002). Biosynthesis of vitamin B2. Eur J Biochem 269(21): 5264-5270.
  5. Somerville, J. E. and Kahn, M. L. (1983). Cloning of the glutamine synthetase I gene from Rhizobium meliloti. J Bacteriol 156(1): 168-176.
  6. Yurgel, S. N., Rice, J., Domreis, E., Lynch, J., Sa, N., Qamar, Z., Rajamani, S., Gao, M., Roje, S. and Bauer, W. D. (2014). Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein. Mol Plant Microbe Interact 27(5): 437-445.

简介

核黄素是黄素核苷酸FMN和FAD的前体,它们在所有生物体中起重要作用。 GTP是核黄素生物合成途径的最初前体,GTP环化水解酶II催化该途径的第一步。 它将GTP转化为2,5-二氨基-6-核糖基氨基-4(3 H) - 嘧啶酮5'-磷酸。 该方案提供了从粗细菌提取物中测定GTP环化水解酶II活性的可靠且快速的方法。 由GTP环化水解酶II 2,5-二氨基-6-核糖基氨基-4(3 H) - 嘧啶酮5'-磷酸催化的反应产物转化为其荧光衍生物6,7- 二甲基蝶呤,然后在XTerra MS C18柱上分离,并使用荧光HPLC系统检测。

材料和试剂

  1. ( 1021,Galibert et al ,2001)
    注意:该协议也可应用于其他细菌。
  2. Bio-Rad蛋白测定试剂盒I(Sigma-Aldrich,目录号:500-0001)
  3. 50mM Tris-HCl缓冲液(pH7.5)(每个样品5ml)
  4. 1x BugBuster试剂(Novagen,目录号:70921)
  5. 0.5 M EDTA(pH 8.0)(每个样品2.5μl)
  6. GTP(鸟苷5'-三磷酸钠盐水合物)(Sigma-Aldrich,目录号:G8877)
  7. HPLC标准:6,7-二甲基蝶呤(Schircks Laboratories,目录号:11.503)
  8. 生物素
  9. YMB培养基(Somerville和Kahn,1983)(参见Recipes)(每个样品1个平板)
  10. MMNH 4介质(Somerville和Kahn,1983)(参见Recipes)(每个样品13ml)
  11. 裂解缓冲液(见配方)(每个样品1ml)
  12. 脱盐缓冲液(见配方)(每个样品3ml)
  13. RibA测定缓冲液(参见Recipes)(每个样品2.5μl)
  14. 衍生试剂(参见配方)(每个样品50μl)
  15. HPLC流动相(参见配方)
    注意:除非另有说明,所有其他化学品均获自Sigma-Aldrich。

设备

  1. 14ml Falcon管(BD Biosciences,目录号:352059)
  2. Eppendorf 1.7ml管(Thermo Fisher Scientific,目录号:14-222-168)
  3. 0.22μm注射器过滤器(Microsolv Technology,目录号:58022-N04-C)
  4. 2ml Zeba Spin Desalting Columns(Thermo Fisher Scientific,目录号:87768)
  5. 微型离心机
  6. 离心机
  7. 振动器
  8. HPLC:Waters Alliance 2695 HPLC系统连接到2475荧光检测器
  9. XTerra MS C18柱(4.6×100mm,5μm)(Waters,部件号:186000486)

程序

  1. 细胞提取物制备
    1. 成长。 meliloti在YMB板上在30℃下培养48小时。 S的最佳温度。 meliloti生长在28℃和30℃之间。
    2. 接种 S. meliloti从原液YMB平板至在3ml MMNH 4培养基中的OD 600约0.1。
    3. 生长细胞在30℃下48小时,250 rpm在14毫升Falcon或玻璃管
    4. 将细胞稀释20倍至10ml新鲜MMNH 4培养基中,并在50ml玻璃管或烧瓶中在30℃,250rpm下生长过夜。  
    5. 通过在3,600×g下在4ml Falcon管中在4℃下离心15-20分钟从10ml培养物收获细胞。  
    6. 弃去上清液并用5 ml 50 mM Tris缓冲液在4°C洗涤细胞
    7. 在4℃下将沉淀重悬在1ml裂解缓冲液中。
    8. 在室温下孵育15分钟,无振荡
    9. 在4℃以最大速度离心裂解液15分钟。
    10. 使用Bio-Rad蛋白质测定试剂盒测量细胞裂解物中的蛋白质浓度
  2. 酶测定
    该测定方案改造自用于测定纯化的酶中的GTP环化水解酶II活性的方法(Bacher等人,1997),并且修改用于细菌提取物。
    1. 按照制造商的说明书,使用2ml Zeba Spin脱盐柱对细胞裂解液进行脱盐。 将柱用3ml脱盐缓冲液平衡。
    2. 加入25μl的总体积为50μl的反应混合物中含有10μl5×RibA测定缓冲液,5μl10mM GTP(在超纯水中制备GTP)的上述步骤的脱盐全细胞裂解物。
    3. 孵育测定在37℃下30分钟
    4. 通过加入2.5μlEDTA(0.5M,pH 8.0)终止测定
    5. 通过加入50μl衍生化试剂,然后在70℃孵育20分钟衍生化产物。
    6. 通过在4℃下以3000xg离心10分钟来清除样品
    7. 使用0.22μm注射器过滤器过滤上清液。
    8. 使用具有荧光检测的HPLC分析衍生化产物
  3. HPLC纯化和信号检测
    1. 使用连接到2475荧光检测器的Waters Alliance 2695 HPLC系统在XTerra MS C18柱(4.6×100mm,5μm)上分离荧光产物。 激发和发射波长分别为330nm和435nm。

  4. 数据分析
    1. 通过与标准比较来量化产品。 通过在HPLC上连续稀释6,7-二甲基蝶呤(0.25nM,2.5nM,25nM,0.25μm,2.5μM和25μM)获得标准曲线。
    2. 归一化酶活性对蛋白质浓度和测定以nmol/min/mg蛋白质的孵育时间。
    3. 在每次测定中进行三次重复。 数据为平均值±S.E。 的三次重复 注意:GTP环化水解酶II的活性在不同生物体中不同。 已经报道纯化的重组酶的活性为约0.1-120nmol/min/mg蛋白质,细菌提取物中的活性可以更低(Herz等人,2000; Kaiser等人,2002; Yurgel等人,2014 )。

代表数据





图1.2.5μM6,7-二甲基蝶呤标准物(A)的HPLC色谱图,来自细菌提取物的GTP环化水解酶II测定的产物(B)和来自GTP环化水解酶II测定的产物与6,7-二甲基蝶呤标准物 )。 HPLC流动相:10%甲醇和90%磷酸(v/v)。 激发和发射波长分别为330nm和435nm。

食谱

  1. YMB媒介根瘤菌

    1 L
    集中
    酵母提取物
    1克

    甘露醇
    10克
    54.9 mM
    Agar
    15克


    高压灭菌,冷却至55°C,然后加入
    YMB盐I
    10 ml
    YMB盐II
    10 ml

    YMB盐I
    1 L
    集中
    K 2 HPO 4
    50克
    287.06 mM
    NaCl
    10克
    171.15 mM
    d H 2 O 2 960 ml


    YMB盐II
    1 L
    集中
    MgSO 4。 。 O
    20克
    81.11 mM
    d H 2 O 2 1 L



  2. MMNH (用于根瘤菌的最小甘露醇氨培养基)

    每1 L
    集中
    甘露醇
    10.0克
    54.9 mM
    NH 4 Cl
    0.5克
    9.34 mM
    琼脂(用于平板制备)
    15.0克

    d H 2 O 2 970 ml


    高压灭菌,冷却至55°C,然后添加:
    生物素(0.2mg/ml,在50%EtOH中) 1.0 ml
    硫胺素(2mg/ml),过滤灭菌
    1.0 ml
    Min Man Salts I
    10.0 ml
    Min Man Salts II
    10.0 ml

    Min Man Salts I

    每1 L
    集中
    K 2 HPO 4
    100克
    574.12 mM
    KH 2 PO 4
    100克
    734.8 mM
    Na 2 4
    25克
    174.8 mM
    d H 2 O 2 1 L

    Min Man Salt II

    每1 L
    集中
    FeCl 3 6H <2> O
    1.0 g
    3.7 mM
    浓HCl
    调节pH至〜7.0(〜1滴)
    CaCl 2 2H O
    10.0克
    68 mM
    MgCl 2 6H <2> O
    25.0克
    123 mM
    d H 2 O 2 1 L
    高压灭菌器

  3. 裂解缓冲液
    50mM Tris-HCl(pH7.5) 10mM MgCl 2/
    1mM三(羟丙基)膦
    1x BugBuster试剂
  4. 脱盐缓冲液
    50mM Tris-HCl(pH7.5) 1mM三(羟丙基)膦
    10%甘油
  5. RibA测定缓冲液
    100mM Tris-HCl(pH8.5)缓冲液
    5mM MgCl 2/
    5mM二硫苏糖醇(DTT)
  6. 衍生试剂
    1%(v/v)二乙酰基 15%(w/v)三氯乙酸
  7. HPLC流动相
    10%(v/v)甲醇
    90%磷酸(27mM磷酸)

致谢

该方案得自Yurgel等人,2014年。该工作得到华盛顿州立大学的农业研究中心(WNP-00773)的支持,并授予DE-FG0396ER20225 Vol。 27,No.5,2014/445,来自美国能源部的能源生物科学计划,并授予NSF-MCB 1052492给S.Rajamani。 这项活动部分资助了华盛顿州立大学农业,人类,自然资源科学和生物密集型农业和有机农业学院农业研究中心的新兴研究问题内部竞争性拨款(BIOAg)内部竞争性拨款 从维持中心 农业和自然资源(CSANR)在华盛顿州立大学S. Yurgel。我们感谢M. Kahn的讨论和华盛顿州立大学生物技术和生物分析实验室的测序支持。

参考文献

  1. Bacher,A.,Richter,G.,Ritz,H.,Eberhardt,S.,Fischer,M。和Krieger,C。(1997)。 核黄素的生物合成:GTP环化水解酶II,脱氨酶和还原酶方法Enzymol 280:382-389。
  2. Galibert,F.,Finan,TM,Long,SR,Puhler,A.,Abola,P.,Ampe,F.,Barloy-Hubler,F.,Barnett,MJ,Becker,A.,Boistard,P.,Bothe G.,Boutry,M.,Bowser,L.,Buhrmester,J.,Cadieu,E.,Capela,D.,Chain,P.,Cowie,A.,Davis,RW,Dreano,S.,Federspiel, NA,Fisher,RF,Gloux,S.,Godrie,T.,Goffeau,A.,Golding,B.,Gouzy,J.,Gurjal,M.,Hernandez-Lucas,I.,Hong,A.,Huizar, L.,Hyman,RW,Jones,T.,Kahn,D.,Kahn,ML,Kalman,S.,Keating,DH,Kiss,E.,Komp,C.,Lelaure,V.,Masuy, Palm,C.,Peck,MC,Pohl,TM,Portetelle,D.,Purnelle,B.,Ramsperger,U.,Surzycki,R.,Thebault,P.,Vandenbol,M.,Vorholter,FJ,Weidner,S 。,Wells,DH,Wong,K.,Yeh,KCand Batut,J。(2001)。 豆科植物共生菌的复合基因组 Sinorhizobium meliloti 。 科学 293(5530):668-672。
  3. Herz,S.,Eberhardt,S。和Bacher,A。(2000)。 核黄素在植物中的生物合成。拟南芥的ribA基因确定了双功能GTP环化水解酶II/3,4-二羟基-2-丁酮4-磷酸合酶。

    53(7) :723-731。
  4. Kaiser,J.,Schramek,N.,Eberhardt,S.,Puttmer,S.,Schuster,M。和Bacher,A。(2002)。 维生素B2的生物合成 Eur J Biochem 269 21):5264-5270。
  5. Somerville,J.E。和Kahn,M.L。(1983)。 克隆来自苜蓿根瘤菌(Rhizobium meliloti)的谷氨酰胺合成酶I基因。 156(1):168-176。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yurgel, S. N., Sa, N., Rice, J. and Roje, S. (2014). Assay for GTP Cyclohydrolase II Activity in Bacterial Extracts. Bio-protocol 4(15): e1198. DOI: 10.21769/BioProtoc.1198.
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