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本实验方案简略版
Jan 2018

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Detection of D-glutamate Production from the Dual Function Enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis
从垢分支杆菌中通过双功能酶,4-氨基-4-脱氧胆固醇裂解酶/D-氨基酸转氨酶、转氨酶产生的D-谷氨酸的检测   

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Abstract

D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis was identified as having D-amino acid transaminase (D-AAT) activity, although it has primarily been annotated as 4-amino-4-deoxychorismate lyase (ADCL). To unequivocally demonstrate D-AAT activity from MSMEG_5795 protein two coupled enzyme assays were performed in series. First, D-alanine and α-ketoglutarate were converted to D-glutamate and pyruvate by MSMEG_5795 using the D-AAT assay. Next, the products of this reaction, following removal of all protein, were used as input into an assay for glutamate racemase in which D-glutamate is converted to L-glutamate by glutamate racemase (Gallo and Knowles, 1993; Poen et al., 2016). As the only source of D-glutamate in this assay would be from the reaction of D-alanine with MSMEG_5795, positive results from this assay would confirm the D-AAT activity of MSMEG_5795 and of any enzyme tested in this manner.

Keywords: D-amino acid transaminase (D-氨基酸转氨酶), Enzyme assay (酶活测), Glutamate racemase (谷氨酸消旋酶), Transaminase (转氨酶), Amino-deoxychorismate lyase (氨基脱氧胆酸裂解酶), Pyridoxal phosphate (磷酸吡哆醛)

Background

The protocol described here in detail was developed during a study of suppressor mutants in M. smegmatis strain in which glutamate racemase (murI) had been deleted (Mortuza et al., 2018). During this work, it became apparent that a mutation in the promoter of 4-amino-4-deoxychorismate lyase (ADCL) (MSMEG_5795) was unexpectedly able to complement the murI deletion. This mutation resulted in a more than 10-fold increased expression of MSMEG_5795. Because ADCL is homologous to D-amino acid transaminase (D-AAT), we decided to test MSMEG_5795 for D-AAT activity. The initial test which involved incubation of the enzyme with D-alanine and α-ketoglutarate was positive for activity. To be doubly sure that this protein, which was predominantly annotated as ADCL, was actually producing glutamate, we used the output of the D-AAT assay as input to the previously reported coupled assay for MurI (Tanizawa et al., 1987; Gallo and Knowles, 1993). This assay is detailed below.

Materials and Reagents

  1. Pipette tips (Labcon, 1,250 µl, catalog number: 1045-260-300; 200 µl, catalog number: 1030-260-300; 10 µl, catalog number: 1036-260-000) 
  2. Microcentrifuge tubes 1.7 ml (MultiMax, catalog number: 2942)
  3. 50 ml conical centrifuge tubes (Cellstar, catalog number: 227261)
  4. Cuvette Type 9 quartz 10 mm path (Starna, catalog number: 9Q10)
  5. VivaspinTM 2 protein concentrator spin column, MWCO 10,000 Da (GE Healthcare Life Sciences, catalog number: 28932247)
  6. Syringe filter, 0.45 µm (Ahlstrom, catalog number: 760506) 
  7. Syringe with Luer-Lok tip, 20 ml (Becton Dickinson, catalog number: 3028360) 
  8. 10-15 mg/ml of purified Mycobacterium smegmatis MSMEG_5795 in 20 mM Tris-HCl pH 8.0 and 150 mM NaCl (Mortuza et al., 2018), and 20-40 mg/ml of purified Bacillus anthracis glutamate racemase isoform 2 (MurI2Ba) protein in 50 mM Tris pH 8.0, 250 mM NaCl and 0.1 mM DTT (May et al., 2007)
  9. Tris base (NeoFroxx, catalog number: 1125KG001)
  10. HCl, 37%, reagent grade, ACS (Scharlau, catalog number: AC07412500)
  11. α-ketoglutaric acid sodium salt (Sigma-Aldrich, catalog number: K1875)
  12. Pyridoxal 5′-phosphate hydrate (PLP) (Sigma-Aldrich, catalog number: P9255)
  13. L-lactate dehydrogenase from hog muscle (Roche, catalog number: 10107085001)
  14. β-Nicotinamide adenine dinucleotide, reduced dipotassium salt (NADH) (Sigma-Aldrich, catalog number: N4505)
  15. D-alanine (Sigma-Aldrich, catalog number: A7377)
  16. NaOH (ACS reagent grade, catalog number: SO042500) 
  17. N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) (ACROS, catalog number: 208181000)
  18. Iodonitrotetrazolium chloride (INT) (Sigma-Aldrich, catalog number: I10406)
  19. Diaphorase (Sigma-Aldrich, catalog number: D5540)
  20. Nicotinamide adenine dinucleotide (NAD+) (Sigma-Aldrich, catalog number: N1511)
  21. Adenosine 5’-diphosphate (ADP) (Sigma-Aldrich, catalog number: A2754)
  22. L-glutamate dehydrogenase (LGDH) (Sigma-Aldrich, catalog number: G2501)
  23. PierceTM Coomassie Plus (Bradford) Assay Kit (Thermo Fisher, catalog number: 23236)
  24. 10 M NaOH (made by gradually dissolving NaOH pellets in ASTM Type I (18 MΩ) water, stored at room temperature, no need to sterilize)

Note: All solutions below are prepared in ASTM Type I (18 MΩ) water and filtered through 0.45 μm filters prior to use. Solutions are stored long-term at 4 °C, unless otherwise noted.

  1. 1 M Tris base (adjust the solution to pH 8.1 with HCl)
  2. 0.5 M α-ketoglutaric acid 
  3. 2 mM PLP
  4. 10 mM NADH (prepare this solution fresh for each assay)
  5. 0.5 M D-alanine
  6. 1 M CHES (adjust the solution to pH 9.2 with NaOH) 
  7. D-amino acid transaminase reaction partial mixture (see Recipes)
  8. Glutamate reaction partial mixture (see Recipes)

Equipment

  1. Magnetic stir bars 15 mm x 6 mm (BRAND, catalog number: 137114)
  2. Pipetman Neo® Pipets (Gilson, model: P1000N, catalog number: F144566; P200N, catalog number: F144565; P20N, catalog number: F144563) 
  3. 25 ml glass beaker (Boeco, catalog number: BOE 5010614)
  4. Microcentrifuge (Eppendorf, model: 5415R)
  5. Magnetic stirrer (Barnstead Thermolyne, model: SP131010-33)
  6. UltrospecTM 3100 pro UV/Visible Spectrophotometer with SWIFT II software (GE Healthcare Life Sciences, model: UltrospecTM 3100 pro)
  7. MultiTemp III (GE Healthcare) thermostatic circulator to control the temperature of the UV-Vis spectrophotometer cuvettes
  8. -20 °C freezer

Software

  1. BiochromTM SWIFT II Software, version 2.05

Procedure

  1. Assay Preparations
    1. Purify 10-15 mg/ml MSMEG_5795 and 20-40 mg/ml MurI2Ba protein using immobilized metal affinity chromatography and size exclusion chromatography.
      Note: Determine the mycobacterial protein concentration using the PierceTM Coomassie Plus (Bradford) Assay according to the manufacturer’s recommendation. Centrifuge both proteins at 16,000 x g for 20 min at 4 °C to remove any aggregates prior to being assayed. 
    2. Prepare all stock solutions for the assays (see Recipes).

  2. D-amino acid transaminase reaction
    1. Prepare desired volume of the D-AAT assay reaction partial mixture containing 100 mM Tris-HCl pH 8.1, 10 mM α-ketoglutarate, 0.15 mM PLP, 5 U L-lactate dehydrogenase, 0.2 mM NADH and 77 nM MSMEG_5795 protein. For example, 20 ml is a convenient amount to make here. As noted above the NADH is prepared fresh each day before conducting the assay.
      Note: Keep assay mix on ice during assay and equilibrate to room temperature before use. 
    2. Transfer 950 µl of the reaction mixture to a 1 ml quartz cuvette and pre-incubate at 30 °C for 5 min.
    3. Measure the absorbance at 340 nm for 2 min to set a baseline on the UltrospecTM 3100 pro. 
    4. To start the reaction, add 50 µl 0.5 M D-alanine substrate to the assay mix to a final concentration of 25 mM to start the reaction and incubate each cuvette for 20, 40 and 60 min at 30 °C. The absorbance should be decreasing with time.
    5. Add 50 µl water instead of substrate to the negative control sample and incubate for 60 min at 30 °C. 
    6. Following incubation, transfer the cuvettes immediately to 4 °C to halt the reaction. 
    7. Filter the reaction mix using a Vivaspin 2 protein concentrator spin column with MWCO of 10,000 Da to remove MSMEG_5795 and L-lactate dehydrogenase. Use this protein-free output as a substrate for the subsequent glutamate racemase (MurI) activity assay.
      Note: Samples can be stored at -20 °C before second coupled enzyme assay is performed or for further analysis. 

  3. Glutamate racemase reaction
    1. Prepare desired volume of MurI assay reaction partial mixture containing 50 mM CHES pH 9.2, 5 mM NAD+, 37.5 U ml-1 L-glutamate dehydrogenase, 2.5 mM ADP, 0.65 mM INT, 2 U ml-1 diaphorase. For example, 10 ml is a convenient amount to make here.
      Note: Mix assay components vigorously in a glass beaker, but avoid foaming by using a small stir bar and magnetic stirrer. Filter mixture with a 0.45 µm syringe filter and a 20 ml syringe with a Luer-Lok tip. Keep solution on ice during assay. Equilibrate to room temperature before use. This solution will take on a light salmon hue.
    2. For each reaction sample, transfer 875 µl of the reaction mixture to a 1 ml quartz cuvette. 
    3. Add 100 µl of the product coming from the D-AAT assay to each cuvette. 
    4. Start the reaction by adding 25 µl MurI2Ba to a final enzyme concentration of 10 µM. 
    5. To the blank sample, add the buffer used for the MurI2Ba stock solution in place of MurI2Ba and add water instead of substrate. 
    6. Add water instead of substrate to the negative control sample. 
    7. Monitor the absorbance at 500 nm for 30 min at 30 °C.
      Note: Subtract the absorbance values obtained from the blank sample from each measurement to obtain the final absorbance values.

Data analysis

The product of the D-AAT assay is taken as input for the glutamate racemase assay to confirm the presence of glutamate by monitoring the absorbance increase at 500 nm. The increase in absorbance values for the 20, 40 and 60 min samples corresponds to the expected increase in glutamate production over time (Figure 1). The detection of glutamate as reported here is qualitative. This result was also supported with data obtained from high-resolution mass spectrometry of the assay output which showed an increase in glutamate concentration of 0.3, 0.7 and 1.0 mM in samples incubated for 20, 40 and 60 minutes respectively. As a result, the ability to convert D-alanine to D-glutamate by the MSEMG_5795 enzyme can be assessed using the glutamate racemase assay.


Figure 1. Time-dependent glutamate production by MSMEG_5795 verified with glutamate racemase enzyme assay (see Step C7). Protein-free product from the D-AAT assay was used as substrate for the glutamate racemase assay in the D to L direction. The increase in absorbance was observed at 500 nm for 30 min. Each sample was taken in duplicate and contained 100 µl substrate and 10 µM glutamate racemase. Blank sample was subtracted from each measurement and absorbance values were averaged before plotting against time. Absorbance values obtained from the 20, 40 and 60 min samples are shown in green, blue and red respectively. The control sample containing water instead of substrate is shown in black. Figure 1 is originally published in Mortuza et al., 2018. (This figure is reprinted with permission from Molecular Microbiology.)

Notes

  1. The concentrations of the components listed in the assay mixtures represent the desired concentrations to have in place at the start of the assay, but be sure to take into account any dilution that may result from the addition of enzyme or other substance added subsequently.
  2. If there is any concern that L-glutamate might be produced by the first enzyme being tested in this assay an additional control can be added in which the output of the first enzyme is assayed using the enzyme L-glutamate dehydrogenase directly without incubation with MurI. 
  3. Other glutamate racemases that are sufficiently active at 30 °C and pH 9.2 would likely work here as well, but we have only trialed this assay with the MurI2Ba enzyme.

Recipes

  1. D-amino acid transaminase reaction partial mixture
    100 mM Tris-HCl, pH 8.1
    10 mM α-ketoglutarate
    0.15 mM PLP
    5 U L-lactate dehydrogenase
    0.2 mM NADH
    77 nM MSMEG_5795 protein
    Prepare in ASTM Type I (18 MΩ) water
    Filter through a 0.45 μm filter prior to use
    Long-term storage not recommended. Prepare fresh each day
    Substrate is added later
  2. Glutamate reaction partial mixture
    50 mM CHES, pH 9.2
    5 mM NAD+
    37.5 U ml-1 L-glutamate dehydrogenase
    2.5 mM ADP
    0.65 mM iodonitrotetrazolium chloride
    2 U ml-1 diaphorase
    Prepare in ASTM Type I (18 MΩ) water
    Filter through a 0.45 μm filter prior to use
    Long-term storage not recommended. Prepare fresh each day
    Substrate and MurI are added later

Acknowledgments

This protocol was first described in brief in Mortuza et al. (2018). This research was supported by Lottery Health Research New Zealand-LHR2017-48905, the University of Otago, the Maurice Wilkins Centre-University of Auckland and the Thrash Foundation, Houston, Texas.

Competing interests

The authors have no conflicts to declare.

References

  1. Gallo, K. A. and Knowles, J. R. (1993). Purification, cloning, and cofactor independence of glutamate racemase from Lactobacillus. Biochemistry 32(15): 3981-3990.
  2. May, M., Mehboob, S., Mulhearn, D. C., Wang, Z., Yu, H., Thatcher, G. R., Santarsiero, B. D., Johnson, M. E. and Mesecar, A. D. (2007). Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design. J Mol Biol 371(5): 1219-1237.
  3. Mortuza, R., Aung, H. L., Taiaroa, G., Opel-Reading, H. K., Kleffmann, T., Cook, G. M. and Krause, K. L. (2018). Overexpression of a newly identified D-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion. Mol Microbiol 107(2): 198-213.
  4. Poen, S., Nakatani, Y., Opel-Reading, H. K., Lasse, M., Dobson, R. C. and Krause, K. L. (2016). Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery. Biochem J 473(9): 1267-1280.
  5. Tanizawa, K., Masu, Y., Asano, S., Tanaka, H. and Soda, K. (1987). D-Amino acid aminotransferase from a thermophile, YM-1: enzymological properties, cloning of the amino acid sequence. In: The 7th International Congress on Chemical and Biological Aspects of Vitamin B6 Catalysis. In: Korpela, T. and Christen, P. (Eds). Turku, Finland Birkhauser Verlag: 43-46.

简介

根据以下反应,D-氨基酸转氨酶(D-AAT)能够合成D-谷氨酸和D-丙氨酸:D-丙氨酸+α-酮戊二酸+ D-谷氨酸+丙酮酸。这两种D-氨基酸是肽聚糖细菌层的必要组分。在我们最近发表的研究中,来自耻垢分枝杆菌的MSMEG_5795被鉴定为具有D-氨基酸转氨酶(D-AAT)活性,尽管它主要注释为4-氨基-4-脱氧胆酸裂解酶(ADCL) )。为了明确证明来自MSMEG_5795蛋白的D-AAT活性,两个偶联的酶测定是连续进行的。首先,使用D-AAT测定,通过MSMEG_5795将D-丙氨酸和α-酮戊二酸转化为D-谷氨酸和丙酮酸。接下来,在除去所有蛋白质后,该反应的产物被用作谷氨酸消旋酶测定的输入,其中D-谷氨酸通过谷氨酸消旋酶转化为L-谷氨酸(Gallo和Knowles,1993; Poen et al。,2016)。由于该测定中D-谷氨酸的唯一来源是来自D-丙氨酸与MSMEG_5795的反应,该测定的阳性结果将证实MSMEG_5795和以这种方式测试的任何酶的D-AAT活性。
【背景】这里详细描述的方案是在 M中的抑制突变体的研究期间开发的。耻垢菌菌株,其中谷氨酸消旋酶( murI )已被删除(Mortuza et al。,2018)。在这项工作中,显而易见的是,4-氨基-4-脱氧胆酸裂解酶(ADCL)(MSMEG_5795)的启动子中的突变出乎意料地能够补充 murI 缺失。该突变导致MSMEG_5795表达增加超过10倍。因为ADCL与D-氨基酸转氨酶(D-AAT)同源,我们决定测试MSMEG_5795的D-AAT活性。涉及将酶与D-丙氨酸和α-酮戊二酸温育的初始试验对活性是阳性的。为了更加确定这种主要注释为ADCL的蛋白质实际上是产生谷氨酸,我们使用D-AAT测定的输出作为先前报道的MurI偶联测定的输入(Tanizawa et al。,1987; Gallo和Knowles,1993)。该测定详述如下。

关键字:D-氨基酸转氨酶, 酶活测, 谷氨酸消旋酶, 转氨酶, 氨基脱氧胆酸裂解酶, 磷酸吡哆醛

材料和试剂

  1. 移液器吸头(Labcon,1,250μl,目录号:1045-260-300;200μl,目录号:1030-260-300;10μl,目录号:1036-260-000) 
  2. 微量离心管1.7 ml(MultiMax,目录号:2942)
  3. 50毫升锥形离心管(Cellstar,目录号:227261)
  4. 比色皿9型石英10毫米路径(Starna,目录号:9Q10)
  5. Vivaspin TM 2蛋白浓缩物旋转柱,MWCO 10,000 Da(GE Healthcare Life Sciences,目录号:28932247)
  6. 注射器过滤器,0.45μm(Ahlstrom,目录号:760506) 
  7. 带Luer-Lok尖端的注射器,20毫升(Becton Dickinson,目录号:3028360) 
  8. 10-15mg / ml纯化的耻垢分枝杆菌 MSMEG_5795,在20mM Tris-HCl pH 8.0和150mM NaCl(Mortuza 等人,2018)和20-40中。在50 mM Tris pH 8.0,250 mM NaCl和0.1 mM中纯化的炭疽芽孢杆菌谷氨酸消旋酶同工型2(MurI2 Ba )蛋白质mg / ml DTT(May et al。,2007)
  9. Tris base(NeoFroxx,目录号:1125KG001)
  10. HCl,37%,试剂级,ACS(Scharlau,目录号:AC07412500)
  11. α-酮戊二酸钠盐(Sigma-Aldrich,目录号:K1875)
  12. 吡哆醛5'-磷酸盐水合物(PLP)(Sigma-Aldrich,目录号:P9255)
  13. 来自猪肌肉的L-乳酸脱氢酶(Roche,目录号:10107085001)
  14. β-烟酰胺腺嘌呤二核苷酸,还原二钾盐(NADH)(西格玛奥德里奇,目录号:N4505)
  15. D-丙氨酸(Sigma-Aldrich,目录号:A7377)
  16. NaOH(ACS试剂等级,目录号:SO042500) 
  17. N-环己基-2-氨基乙磺酸(CHES)(ACROS,目录号:208181000)
  18. Iodonitrotetrazolium chloride(INT)(西格玛奥德里奇,目录号:I10406)
  19. Diaphorase(西格玛奥德里奇,目录号:D5540)
  20. 烟酰胺腺嘌呤二核苷酸(NAD + )(Sigma-Aldrich,目录号:N1511)
  21. 腺苷5'-二磷酸(ADP)(Sigma-Aldrich,目录号:A2754)
  22. L-谷氨酸脱氢酶(LGDH)(西格玛奥德里奇,目录号:G2501)
  23. Pierce TM Coomassie Plus(Bradford)检测试剂盒(Thermo Fisher,目录号:23236)
  24. 10 M NaOH(通过在ASTM I型(18MΩ)水中逐渐溶解NaOH颗粒制成,在室温下储存,无需消毒)

注意:以下所有溶液均采用ASTM I型(18MΩ)水制备,并在使用前通过0.45μm过滤器过滤。除非另有说明,否则溶液长期储存在4°C。

  1. 1 M Tris碱(用HCl调节溶液至pH 8.1)
  2. 0.5Mα-酮戊二酸 
  3. 2 mM PLP
  4. 10 mM NADH(为每次检测准备新溶液)
  5. 0.5 M D-丙氨酸
  6. 1 M CHES(用NaOH调节溶液至pH 9.2) 
  7. D-氨基酸转氨酶反应部分混合物(见食谱)
  8. 谷氨酸反应部分混合物(见食谱)

设备

  1. 磁力搅拌棒15 mm x 6 mm(BRAND,目录号:137114)
  2. Pipetman Neo ® Pipets(Gilson,型号:P1000N,目录号:F144566; P200N,目录号:F144565; P20N,目录号:F144563) 
  3. 25毫升玻璃烧杯(Boeco,目录号:BOE 5010614)
  4. 微量离心机(Eppendorf,型号:5415R)
  5. 磁力搅拌器(Barnstead Thermolyne,型号:SP131010-33)
  6. Ultrospec TM 3100 pro紫外/可见分光光度计,带SWIFT II软件(GE Healthcare Life Sciences,型号:Ultrospec TM 3100 pro)
  7. MultiTemp III(GE Healthcare)恒温循环器控制紫外 - 可见分光光度计比色皿的温度
  8. -20°C冰柜

软件

  1. Biochrom TM SWIFT II软件,版本2.05

程序

  1. 分析准备工作
    1. 使用固定化金属亲和层析和尺寸排阻色谱纯化10-15 mg / ml MSMEG_5795和20-40 mg / ml MurI2 Ba 蛋白。
      注意:根据制造商的建议,使用Pierce TM Coomassie Plus(Bradford)Assay测定分枝杆菌蛋白质浓度。在4°C下将两种蛋白质以16,000 x g离心20分钟,以在分析前除去任何聚集物。
    2. 准备所有用于分析的储备溶液(参见食谱)。

  2. D-氨基酸转氨酶反应
    1. 制备所需体积的含有100mM Tris-HCl pH8.1,10mMα-酮戊二酸,0.15mM PLP,5U L-乳酸脱氢酶,0.2mM NADH和77nM MSMEG_5795蛋白的D-AAT测定反应部分混合物。例如,20毫升是这里方便的量。如上所述,在进行测定之前,每天新鲜制备NADH。
      注意:在测定过程中将测定混合物保存在冰上并在使用前平衡至室温。
    2. 将950μl反应混合物转移至1ml石英比色杯中,并在30℃下预孵育5分钟。
    3. 测量340nm处的吸光度2分钟,以在Ultrospec TM 3100 pro上设置基线。 
    4. 为了开始反应,向测定混合物中加入50μl0.5MD-丙氨酸底物至终浓度为25mM以开始反应并在30℃下孵育每个比色杯20,40和60分钟。吸光度应随时间降低。
    5. 向阴性对照样品中加入50μl水代替底物,并在30℃下孵育60分钟。 
    6. 孵育后,立即将比色皿转移至4°C以停止反应。 
    7. 使用具有10,000Da MWCO的Vivaspin 2蛋白质浓缩器旋转柱过滤反应混合物以除去MSMEG_5795和L-乳酸脱氢酶。使用这种无蛋白质输出作为随后的谷氨酸消旋酶(MurI)活性测定的底物。
      注意:在进行第二次偶联酶测定或进行进一步分析之前,样品可以在-20°C下储存。

  3. 谷氨酸消旋酶反应
    1. 制备所需体积的MurI测定反应部分混合物,其含有50mM CHES pH 9.2,5mM NAD + ,37.5U ml -1 L-谷氨酸脱氢酶,2.5mM ADP,0.65 mM INT,2 U ml -1 心肌黄酶。例如,10毫升是这里方便的量。
      注意:在玻璃烧杯中剧烈混合测定组分,但使用小搅拌棒和磁力搅拌器避免发泡。用0.45μm注射器过滤器和带有Luer-Lok尖端的20ml注射器过滤混合物。在测定期间将溶液保持在冰上。使用前平衡至室温。这种解决方案将采用淡淡的鲑鱼色调。
    2. 对于每个反应样品,将875μl反应混合物转移到1 ml石英比色皿中。 
    3. 将来自D-AAT测定的100μl产物加入每个比色杯中。 
    4. 通过添加25μlMurI2 Ba 至最终酶浓度为10μM开始反应。 
    5. 在空白样品中,添加用于MurI2 Ba 原液的缓冲液代替MurI2 Ba 并添加水代替基质。 
    6. 在阴性对照样品中加入水而不是底物。 
    7. 在30℃下监测500nm处的吸光度30分钟。
      注意:从每次测量中减去从空白样品中获得的吸光度值,以获得最终的吸光度值。

数据分析

将D-AAT测定的产物作为谷氨酸消旋酶测定的输入,通过监测500nm处的吸光度增加来确认谷氨酸的存在。 20,40和60分钟样品的吸光度值的增加对应于谷氨酸产生随时间的预期增加(图1)。这里报道的谷氨酸检测是定性的。从分析输出的高分辨率质谱法获得的数据也支持该结果,其显示在分别孵育20,40和60分钟的样品中谷氨酸浓度增加0.3,0.7和1.0mM。结果,可以使用谷氨酸消旋酶测定评估MSEMG_5795酶将D-丙氨酸转化为D-谷氨酸的能力。


图1.通过谷氨酸消旋酶测定验证MSMEG_5795的时间依赖性谷氨酸产生(参见步骤C7)。 来自D-AAT测定的无蛋白产物用作D-L方向的谷氨酸消旋酶测定的底物。在500nm处观察到吸光度的增加30分钟。每个样品一式两份并含有100μl底物和10μM谷氨酸消旋酶。从每次测量中减去空白样品,并在绘制时间之前平均吸光度值。从20,40和60分钟样品获得的吸光度值分别以绿色,蓝色和红色显示。含有水而不是底物的对照样品显示为黑色。图1最初发表于Mortuza et al。,2018。(该图经分子微生物学许可转载。)

笔记

  1. 测定混合物中列出的组分浓度代表在测定开始时所需的浓度,但一定要考虑加入随后加入的酶或其他物质可能导致的任何稀释。
  2. 如果担心在该测定中可能由第一种酶产生L-谷氨酸,则可以加入另外的对照,其中使用酶L-谷氨酸脱氢酶直接测定第一种酶的输出而不与MurI孵育。  
  3. 其他在30°C和pH 9.2下具有足够活性的谷氨酸消旋酶也可能在这里起作用,但我们只用MurI2 Ba 酶试验了这种测定。 />

食谱

  1. D-氨基酸转氨酶反应部分混合物
    100mM Tris-HCl,pH 8.1
    10mMα-酮戊二酸
    0.15 mM PLP
    5 U乳酸脱氢酶
    0.2 mM NADH
    77 nM MSMEG_5795蛋白质
    用ASTM I型(18MΩ)水制备
    使用前通过0.45μm过滤器过滤
    不建议长期存放。每天准备新鲜
    稍后添加底物
  2. 谷氨酸反应部分混合物
    50 mM CHES,pH 9.2
    5 mM NAD +
    37.5U ml -1 L-谷氨酸脱氢酶
    2.5 mM ADP
    0.65 mM碘硝基四唑氯化物
    2 U ml -1 心肌黄酶
    用ASTM I型(18MΩ)水制备
    使用前通过0.45μm过滤器过滤
    不建议长期存放。每天准备新鲜
    Substrate和MurI稍后加入

致谢

该方案首先在Mortuza 等人的文章中简要描述(2018)。该研究得到了新西兰彩票健康研究-LHR2017-48905,奥塔哥大学,莫斯科威尔金斯中心 - 奥克兰大学和德克萨斯州休斯顿的Thrash基金会的支持。

利益争夺

作者没有冲突要宣布。

参考

  1. Gallo,K。A.和Knowles,J.R。(1993)。 来自 Lactobacillus 的谷氨酸消旋酶的纯化,克隆和辅因子独立性。< / a> 生物化学 32(15):3981-3990。
  2. May,M.,Mehboob,S.,Mulhearn,D.C.,Wang,Z.,Yu,H.,Thatcher,G.R.,Santarsiero,B.D.,Johnson,M.E。和Mesecar,A.D。(2007)。 来自炭疽芽孢杆菌的两种谷氨酸消旋酶同工酶的结构和功能分析及其影响抑制剂设计。 J Mol Biol 371(5):1219-1237。
  3. Mortuza,R.,Aung,H.L.,Taiaroa,G.,Opel-Reading,H.K.,Kleffmann,T.,Cook,G.M。和Krause,K.L。(2018)。 耻垢分枝杆菌补体中新鉴定的D-氨基酸转氨酶的过表达谷氨酸消旋酶缺失。 Mol Microbiol 107(2):198-213。
  4. Poen,S.,Nakatani,Y.,Opel-Reading,H.K.,Lasse,M.,Dobson,R。C. and Krause,K.L。(2016)。 E 探索结核分枝杆菌的谷氨酸消旋酶结构作为模板抗分枝杆菌药物的发现。 Biochem J 473(9):1267-1280。
  5. Tanizawa,K.,Masu,Y.,Asano,S.,Tanaka,H。和Soda,K。(1987)。 来自嗜热菌的D-氨基酸氨基转移酶,YM-1:酶学特性,克隆氨基酸序列。在:第七届国际化学和生物学方面的维生素B6催化。在:Korpela,T。和Christen,P。(编辑)。芬兰图尔库Birkhauser Verlag:43-46。
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引用:Opel-Reading, H. K., Mortuza, R. and Krause, K. L. (2019). Detection of D-glutamate Production from the Dual Function Enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis. Bio-protocol 9(1): e3135. DOI: 10.21769/BioProtoc.3135.
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