Department of Biochemistry, University of Toronto
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Kanjee, U., Houry, W. A. An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase. J. Vis. Exp. (46), e2094, doi:10.3791/2094 (2010).
Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 Å, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the α-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N′-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.
1) Reagents and Equipment
2) LdcI Assay
3) TNBS Reaction and Color Development
4) Representative Results
1)Cadaverine Standard Curve (Figure 3)
The assay was performed as described but without any L-lysine or LdcI and instead various concentrations of cadaverine were used to empirically determine the linear range of the assay. The assay was linear to an OD340 of 0.25, corresponding to ~ 22 nmoles of cadaverine (Figure 3).
2)Activity of LdcI (Figure 4)
The activity of LdcI alone was determined to be 153.5 (± 18.1) nmoles cadaverine min-1 μg LdcI-1 at pH 6.5. The activity of LdcI is unaffected in the presence of 100 μM GTP or GDP but is strongly inhibited (>10-fold) in the presence of pppGpp and ppGpp (Figure 4).
Figure 1. Schematic diagram of the LdcI reaction. The decarboxylation reaction of L-lysine to generate CO2 and cadaverine is shown as well as the subsequent reaction with TNBS at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N'-bistrinitrophenyllysine (TNP-lysine). Based on Phan et al.11
Figure 2. Setup of Eppendorf Thermostat. The layout of the samples in the 24-well Eppendorf ThermoStat is shown along with description of the steps of the assay (indicated in bold). Arrows indicate the transfer of reaction solutions according to the protocol. Removal of the reaction solution to the stop solution in the 96-well plate is also indicated.
Figure 3. Cadaverine Standard Curve. A plot of absorbance at 340 nm versus nmoles of cadaverine is shown. Error bars represent the standard deviation of at least three independent measurements. The line of best fit is also shown and has an R2 of 0.996.
Figure 4. LdcI assay results. A plot of the rate of LdcI activity (in nmoles cadaverine produced min-1 μg-1 LdcI) is shown in the presence of 100 μM GTP, GDP, pppGpp, ppGpp and in the absence of nucleotide. The stringent response nucleotides (p)ppGpp are capable of significantly inhibiting LdcI activity. The error bars represent the standard deviation of at least six independent measurements.
In the lysine decarboxylase assay, TNBS is reacted with the primary amines of L-lysine and cadaverine to form TNP-lysine and TNP-cadaverine adducts (Figure 1). Due to the presence of the carboxylic acid group on TNP-lysine, this adduct remains soluble in water while the TNP-cadaverine, lacking the carboxylic acid group, is capable of partitioning into toluene11. This type of assay can be utilized more broadly on other types of amino acids where the loss of a carboxylic acid group occurs during the reaction. This occurs during the decarboxylation of L-ornithine by the inducible ornithine decarboxylase SpeF to form the polyamine putrescine7 and the decarboxylation of L-arginine by the inducible arginine decarboxylase AdiA to form the polyamine agmatine4.
The LdcI assay described here provides a relatively fast method for the determination of the activity of the purified protein in vitro. The major advantages of this assay are:
i)Use of multiple replicates per experiment improves the precision of each measurement;
ii)The assay may be conducted over a wide range of buffer conditions (different pH, salt, reducing agent etc.) without modification of the protocol;
iii)The assay may be modified for measuring the in vivo activity of LdcI by determining the amount of cadaverine excreted during cell growth.
The major limitations of this assay are:
i)The sensitivity of the experiments are limited by the linear range of absorbance of the TNP-adducts;
ii)The multiple processing steps increase the magnitude of experimental errors;
iii)Not all amino acid decarboxylases are amenable to this type of protocol. For example, the decarboxylation of L-glutamic acid by the inducible glutamic acid decarboxylases GadA/GadB generates γ-amino-butyric acid2, the TNBS-adduct of which will be soluble in water due to the presence of the side-chain carboxylic acid group.
The biochemical investigation of the acid stress response of E. coli is an expanding area of research and will allow us to better understand the molecular basis of stress response in E. coli and related γ-proteobacteria that have similar acid stress response systems such as Salmonella enterica serovar Typhimurium12 and Vibrio cholera13. The discovery that LdcI activity is inhibited by the stringent response regulator ppGpp has provided us with a previously unknown insight into the regulation of this protein.
No conflicts of interest declared.
We thank Dr. Dr. Michael Cashel (National Institutes of Health, Bethesda, MA, USA) for sending us bacterial strains, plasmids, and necessary protocols. We thank Dr. John Glover (Department of Biochemistry, University of Toronto) for use of the SpecraMax plate reader. UK is the recipient of a National Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship, a Canadian Institutes of Health Research Strategic Training Program in the Structural Biology of Membrane Proteins Linked to Disease, and a University of Toronto Open Fellowship. This work was supported by a grant from the Canadian Institutes of Health Research (MOP-67210) to WAH.
|0.1 mM pyridoxal 5’-phosphate (PLP)||Sigma-Aldrich||P9255|
|96 well polystyrene plates||Sarstedt Ltd|
|96-well quartz plate||Hellma|
|VWR Digital Heatblock||VWR international|
|2.0 mL 96-well polypropylene plates||Axygen Scientific||P-DW-20-C|
|Handy Step Repeat Pipettor||Brand GmbH|
|12.5 mL Repeat Pipettor Tips||Brand GmbH||702378|
|SpectraMax 340PC Plate Reader||SpectraMax|