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Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases

用于确定短链异戊烯基二磷酸合酶产物谱的非放射性分析

AR Avanish Rai
DN Dinesh A. Nagegowda

发布: 2021年01月05日第11卷第1期 DOI: 10.21769/BioProtoc.3874 浏览次数: 3009

评审: Yu LiuNoelia ForesiAnonymous reviewer(s)

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Abstract

Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed using radiolabelled substrates, and the products formed are identified by employing expensive instruments such as phosphor imager, radio-GC, or radioHPLC. Though a non-radioactive assay for measuring IDS activity in crude plant extract has been reported, it requires a complex methodology utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS). Here, we describe a non-radioactive and simple inexpensive assay for determining the IDS assay products using non-radiolabeled IPP and its co-allylic substrates DMAPP,GPP, and FPP. The detection of prenyl diphosphate products generated in the assay was highly efficient and spots corresponding to prenyl alcohols were visible at >40 µM concentrations of IPP and DMAPP/GPP/FPP substrates. The protocol described here is sensitive, reliable, and technically simple, which could be used for functional characterization of IDS candidates.

Keywords: Isoprenyl diphosphate synthase (异戊烯基二磷酸合酶) search Non-radioactive (非放射性) search Phosphatase (磷酸酶) search Apyrase (腺苷三磷酸双磷酸酶) search Prenyl alcohol (异戊烯醇) search Thin layer chromatography (薄层色谱法) search

Background

Short-chain IDSs catalyze the chain length elongation reaction (1′-4 condensations of IPP units) in which allylic isoprenoids substrates C5-DMAPP, C10-GPP, or C15-FPP couple with IPP to generate linear isoprenoids (Figure 1). For instance, Catharanthus roseus GPPS large subunit (CrGPPS.LSU) and GGPP synthase 2 (CrGGPPS2) catalyze the formation of GPP/GGPP [Figure 4 in Rai et al., 2013] and GGPP [Figure 3 in Kumar et al., 2020], respectively, by sequential incorporation of IPP units with DMAPP, GPP, or FPP as substrates. The most widely used assay for determination of IDS activity is discontinuous, time and labor intensive, and involves the use of IPP radiolabeled with either 14[C] or 3[H]. A non-radioactive and LC–MS-based method for determining IDS activity has been reported using crude plant extract utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS) (Nagel et al., 2012). The protocol described here provides an efficient approach to determine the IDS activity without the need for radioactive substrates and high-end instrumentation. The method involves thin layer chromatography (TLC) separation of IDS assay products and visualization and quantification by iodine staining. The assay products generated can be extracted from TLC plate and further verified through LC/MS-based approach as reported in Kumar et al., 2020. We have used purified CrGPPS.LSU, a bifunctional G(G)PP synthase (EC 2.5.1.29) catalyzing the formation of both GPP and GGPP (Rai et al., 2013) in this protocol. The IDS assay described here is highly specific, sensitive, and technically simple which can be useful in determining the functional activity of short-chain IDS enzymes. The efficiency of enzymatic product detection with this method is comparable to that of previously reported radioactive and non-radioactive assays in terms of substrate concentrations.



Figure 1. IPP and DMAPP are utilized in the formation of GPP (C10), FPP (C15), and GGPP (C20) in a reaction catalyzed by short-chain IDS such as geranyl diphosphate synthases (GPPS), farnesyl diphosphate synthases (FPPS), and geranylgeranyl diphosphate synthase (GGPPS), respectively. While FPPS catalyzes the formation of FPP by condensation of DMAPP with 2 IPP units or GPP with one IPP unit, GGPPS forms of GGPP by condensation of DMAPP with three IPP units, GPP with two IPP units, or FPP with one IPP unit.

Materials and Reagents

Note: The materials and reagents not provided with company and catalog number can be ordered from any qualified company for using in this experiment.

  1. Pipette tips (Axygen, USA)

  2. 1.5 ml Microcentrifuge tubes (Eppendorf, catalog number: T9661 )

  3. Parafilm (Sigma-Aldrich, catalog number: P7793 )

  4. Pencils

  5. Disposable latex gloves

  6. Poly-Prep chromatography columns (Bio-Rad, catalog number: 7311550 )

  7. Disposable PD-10 desalting columns (GE Healthcare, catalog number: GE17-0851-01 )

  8. Purified isoprenyl diphosphate synthase [Catharanthus roseus GPPS large subunit (CrGPPS.LSU) for this protocol]

  9. Rosetta 2(DE3) Competent Cells (Novagen, catalog number: 71400-M )

  10. pET-28a(+) vector (Novagen, catalog number: 69864 )

  11. Ni2+-charged nitrilotriacetic acid (NTA) Agarose (Bio-Rad, catalog number: 7800800 )

  12. Luria Bertani Broth, Miller (HIMEDIA, catalog number: M1245 )

  13. Luria Bertani Agar (HIMEDIA, catalog number: M1151F )

  14. Kanamycin sulfate (Sigma-Aldrich, catalog number: 10106801001 )

  15. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )

  16. Imidazole (Sigma-Aldrich, catalog number: I5513-25G )

  17. Lysozyme from hen egg white (Sigma-Aldrich, catalog number: 10837059001 )

  18. Isopentenyl diphosphate (Echelon Biosciences, catalog number: I-0050 )

  19. Dimethylallyl diphosphate (Echelon Biosciences, catalog number: I-0051 )

  20. Geranyl diphosphate (Echelon Biosciences, catalog number: I-0100 )

  21. Farnesyl diphosphate (Echelon Biosciences, catalog number: I-0150 )

  22. Apyrase from potatoes (Sigma-Aldrich, catalog number: A6132 )

  23. Alkaline phosphatase from calf intestine (CIP) (Sigma-Aldrich, catalog number: P4978 )

  24. Geraniol (Sigma-Aldrich, catalog number: 48798 )

  25. Farnesol (Sigma-Aldrich, catalog number: 43348 )

  26. Geranylgeraniol (Sigma-Aldrich, catalog number: G3278 )

  27. TLC Silica gel 60 RP-18 F254s (Merck, catalog number: 105560 )

  28. Glycine (Sigma-Aldrich, catalog number: G7126-100G )

  29. 10% Mini-PROTEAN® TGXTM precast protein gels (Bio-Rad, catalog number: 4561033 )

  30. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771-100G )

  31. Bradford Protein Assay Dye Reagent (Bio-Rad, catalog number: 5000006 )

  32. Coomassie brilliant blue (CBB-R-250) (Sigma-Aldrich, catalog number: B-7920-10G )

  33. Bromophenol blue (Sigma-Aldrich, catalog number: B5525-5G )

  34. Ethanol (Emsure, catalog number: 1.00983.0511 )

  35. Water HPLC grade (Sigma-Aldrich, catalog number: 270733 )

  36. Glycerol (Sigma-Aldrich, catalog number: G5516 )

  37. 3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO) (Sigma-Aldrich, catalog number: M8389 )

  38. 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10197777001 )

  39. Magnesium chloride (MgCl2) (HIMEDIA, catalog number: GRM4340 )

  40. Iodine (I2) crystals (Sigma-Aldrich, catalog number: 229695 )

  41. n-Hexane (Merck, catalog number: 104369 )

  42. TRIS hydrochloride (Tris-HCl) (HIMEDIA, catalog number: MB030 )

  43. Methanol (Merck, catalog number: 60600905001730 )

  44. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502-5G )

  45. Lysis buffer (see Recipes)

  46. Wash buffer (see Recipes)

  47. Elution buffer (see Recipes)

  48. Storage buffer (see Recipes)

  49. Assay buffer (see Recipes)

  50. Dephosphorylation buffer (see Recipes)

  51. Mobile phase (see Recipes)

  52. TG buffer (see Recipes)

Equipment

Note: The equipment not provided with company and catalog number can be ordered from any qualified company for using in this experiment.

  1. Pipettes

  2. Eppendorf Thermo Mixer® F1.5 (Eppendorf, catalog number: 5384000012 )

  3. Glass rectangular developing chamber 20 x 20 cm (with lid)

  4. Measuring cylinders

  5. Scale ruler

  6. Tweezers

  7. Scissors

  8. Borosilicate beakers, capacity 500 ml

  9. Rocker (GeNei, catalog number: 107106GB )

  10. Variable volume pipettes (Eppendorf)

  11. Magnetic stirrer (GeNei, catalog number: 117795GB )

  12. Adsorbent TLC scraper (Sigma-Aldrich, catalog number: Z265268 )

  13. Vacuum concentrator (Savant Speed Vac, catalog number: SPD131DDA )

  14. Fume hood

  15. Sonicator (PRO Scientific, catalog number: H-1021-2 )

  16. Circulating refrigerated water bath

  17. ThermoMixer C (Eppendorf, catalog number: 5382000015 )

  18. Vortex mixer (GeNei, catalog number: 106887GB )

  19. Centrifuge 5424 R (Eppendorf, catalog number: 5404000014 )

  20. Digital camera or scanner

Software

  1. ImageJ (National Institutes of Health, USA, https://imagej.nih.gov/ij/)

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Rai, A. and Nagegowda, D. (2021). Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases. Bio-protocol 11(1): e3874. DOI: 10.21769/BioProtoc.3874.

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植物科学 > 植物生物化学 > 蛋白质

生物化学 > 蛋白质 > 活性

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