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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

HO Helen K. Opel-Reading
RM Roman Mortuza
KK Kurt L. Krause

发布: 2019年01月05日第9卷第1期 DOI: 10.21769/BioProtoc.3135 浏览次数: 5981

评审: Valentine V TrotterWilliam Ryan WillAnonymous reviewer(s)

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Cover of Molecular Microbiology, featuring study using the protocol.

Jan 2018

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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) search Enzyme assay (酶活测) search Glutamate racemase (谷氨酸消旋酶) search Transaminase (转氨酶) search Amino-deoxychorismate lyase (氨基脱氧胆酸裂解酶) search Pyridoxal phosphate (磷酸吡哆醛) search

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

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© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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微生物学 > 微生物生物化学 > 其它化合物

生物化学 > 其它化合物 > 氨基酸

生物化学 > 蛋白质 > 活性

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