Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

Article abstract of DOI:10.1021/acs.analchem.7b03556

S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5′-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a K_d value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5′-methylthioadenosine, AMP, and ADP.

Full text of DOI:10.1021/acs.analchem.7b03556

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Article
Direct Detection of Products from S-adenosylmethionine-Dependent
Enzymes using a Competitive Fluorescence Polarization Assay
Michael T Banco, Vidhi Mishra, Samantha C. Greeley, and Donald R Ronning
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03556 • Publication Date (Web): 25 Dec 2017
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Direct Detection of Products from S-adenosylmethionine-Dependent Enzymes using
a Competitive Fluorescence Polarization Assay
Michael T. Bancoa, Vidhi Mishraa, Samantha C. Greeleyb and Donald R. Ronning*a
aDepartment of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606
bDepartment of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109
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Fax: (419) 530ꢀ4033
Email: Donald.Ronning@utoledo.edu
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Abstract
Sꢀadenosylmethionine (AdoMet) dependent methyltransferases (MTases) are an essential
superfamily of enzymes that catalyze the transfer of a methyl group to several
biomolecules. Alterations in the methylation of cellular components crucially impact vital
biological processes, making MTases attractive drug targets for treating infectious
diseases and diseases caused by overactive humanꢀencoded MTases. Several methods
have been developed for monitoring the activity of MTases, but most MTase assays have
inherent limitations or are not amenable for highꢀthroughput screening. We describe a
universal, competitive fluorescence polarization (FP) assay that directly measures the
production of Sꢀadenosylhomocysteine (AdoHcy) from MTases. Our developed assay
monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy
molecule complexed to a catalytically inert 5′ꢀmethylthioadenosine nucleosidase
(MTANꢀD198N) variant performed in a mixꢀandꢀread format. Producing the
fluorescently labeled molecule involves a oneꢀpot synthesis by combining AdoHcy with
an amineꢀreactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to
MTANꢀD198N. The developed competitive FP assay expresses a limit of detection for
AdoHcy of 6 nM, and exhibits a 34ꢀfold preference to AdoHcy in comparison to
AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen
with the NIH Clinical Collection as well as determining the kinetic parameters of Lꢀ
histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the
developed assay is applicable to other AdoMetꢀdependent and ATPꢀdependent enzymes
by detecting adenosine and various adenosineꢀcontaining molecules including 5′ꢀ
methylthioadenosine, AMP and ADP.
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INTRODUCTION
Sꢀadenosylmethionine (AdoMet) serves as an essential substrate for a myriad of transfer
reactions in bacterial and eukaryotic cells, which include postꢀtranslational modification
of proteins, epigenetic regulation, polyamine biosynthesis, radical pathways, and quorum
sensing 1,2. In this role, AdoMet is the second most widely used metabolite as a cofactor
for enzymatic reactions following ATP 3. AdoMetꢀdependent methyltransferases
(MTases) are the diverse class of enzymes that assist in the methylation of numerous
biomolecular components in cells from all 3 domains of life. MTases catalyze the transfer
of a methyl group from AdoMet to an acceptor molecule, generating Sꢀ
adenosylhomocysteine (AdoHcy) and the modified methylated molecule. Additionally,
products generated from AdoMetꢀdependent enzymes including AdoHcy, 5′ꢀ
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methylthioadenosine (MTA), and 5′ꢀdeoxyadenosine are known potent inhibitors of
enzymes that catalyze methylation reactions, polyamine biosynthesis and AdoMet radical
pathways, respectively 4ꢀ7. In some bacteria, the cellular concentrations of products from
AdoMetꢀdependent enzymes are catabolized by MTA/AdoHcy nucleosidase (MTAN) to
avoid product inhibition of these vital biochemical processes. Additionally, undesired
methylation by MTases in humans leads to epigenetic dysregulation of transcription,
which is demonstrated to play an important role in carcinogenesis. More specifically,
alterations in the methylation of histones and DNA by MTases result in chromosomal
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instability and spurious gene expression, consequently leading to tumorigenesis
.
Furthermore, methylation of A2058 of 23S rRNA in the large subunit of the Escherichia
coli ribosome by MTases have been shown to promote intrinsic resistant to three distinct
classes of antibiotics including macrolides, lincosamides and quinupristin 10ꢀ12. For these
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reasons, there is great interest in enzymatic characterization of MTase function as well as
development of novel inhibitors for MTases. Such compounds could be used as
antimicrobials or anticancer therapeutics.
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Various methods have been developed to directly and indirectly monitor the activity of
MTases, but these lack sensitivity, exhibit inefficiency in medium to high throughput
screening, or require radioisotopic labeling of substrates. Several of these assays are
dependent on multiple coupled enzymes to process the generated AdoHcy product into a
chromophore or luminophore 13ꢀ15. MTAN is a common auxiliary enzyme used in kinetic
assays for MTases due to the ability to catalyze the hydrolysis of AdoHcy into an adenine
and Sꢀribosylhomocysteine molecule, which assists in eliminating product inhibition for
the AdoMetꢀdependent enzyme. For example, a spectroscopicꢀbased assay developed by
Dorgan and coworkers incorporates adenine deaminase to convert the MTANꢀgenerated
adenine into hypoxanthine to monitor the activity of a MTase 15. Earlyꢀdeveloped
spectroscopic kinetic methods for MTases exhibit inherently low sensitivity when
detecting AdoHcy, which becomes problematic for performing kinetic analyses on
catalytically slow enzymes. Alternatively, luminescenceꢀbased assays developed for
MTases have demonstrated to be highly sensitive for detecting generated AdoHcy 14,16,17
.
For instance, coupling MTAN, adenine phosphoribosyl transferase, and pyruvate
orthophosphate dikinase to produce ATP that is then used by luciferase to generate a
luminescent signal was determined to possess an observed limit of detection (LOD) for
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AdoHcy of 0.1 pmol . An inherent challenge of efficiently screening small molecule
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libraries with coupled enzymes is the elevated hit rate of false positives and the
requirement for orthogonal assays to validate hits 19.
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Assays that directly measure AdoHcy or methylated products improve the adaptability
for highꢀthroughput screening by potentially reducing the hit rate of false positives.
Immunoꢀbased assays that directly detect methylated histone products have been
demonstrated to be feasible for medium to highꢀthroughput screening by successfully
identifying inhibitors. For example, such assays have identified inhibitors targeting a G9a
MTase, which is a eukaryotic histone lysine MTase 20ꢀ22. Conversely, detecting AdoHcy
rather than specific methylated products provides a universal detection mechanism that
can be applied to any MTase. For instance, fluorescence polarization (FP) is a widelyꢀ
used technique that monitors the fractional binding of a fluorescently labeled ligand to an
enzyme. Graves and coꢀworkers previously developed a competitive FP immunoassay for
detecting generated AdoHcy from MTases, which differentiates AdoHcy from
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background AdoMet and exhibits a LOD of 0.5 pmol of AdoHcy . Although direct
immunodetection of AdoHcy provides several advantages for highꢀthroughput screening
of smallꢀmolecule libraries, production of highꢀtiter antibodies can be prohibitively
expensive.
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Figure 1: (A) The chemical structure of AdoHcy-TAMRA. (B) The chemical structures of the various
adenosine-containing molecules that are quantifiable by the HpMTAN-D198N/AdoHcy-TAMRA
competitive FP assay.
Here, we present a generic, homogenous, competitive FP assay that allows for the direct
quantification of AdoHcy as well as several other adenosineꢀcontaining molecules. The
concept for the FP assay involves leveraging a Helicobacter pylori MTAN variant
(HpMTANꢀD198N) that prevents catalytic turnover, while allowing the binding of a
AdoHcy molecule conjugated to a fluorophore on the αꢀamino group of the homocysteine
moiety. The synthesized fluorescent probe demonstrates a Kd value in the low nM range
(AdoHcyꢀTAMRA, Figure 1A), which allows for a wide range of concentrations to be
resolved while performing competitive FP assays 24. The results from our competitive FP
experiments highlight the versatility and sensitivity of measuring various generated
products from AdoMetꢀdependent as well as ATPꢀdependent enzymes, including
AdoHcy, MTA, ADP, and AMP (Figure 1B). Notably, our developed competitive FP
method provides an alternative assay for monitoring the activity of MTases that is highly
sensitive, cost effective, and suitable for highꢀthroughput screening.
EXPERIMENTAL
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Purification of HpMTAN-D198N and Mycobacterium tuberculosis EgtD
The cloning and expression of Helicobacter pylori MTANꢀD198N and Mycobacterium
tuberculosis (Mtb) EgtD were performed as previously stated 25,26. An inꢀdepth protocol
for cloning and purification for both enzymes can be found in the supplemental section of
the manuscript.
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Synthesis and Purification of the AdoHcy fluorescent probe
Oneꢀpot synthesis of the fluorescent probe was performed by combining AdoHcy (1.3
mg, Sigma) in a 100 mM sodium bicarbonate buffer at pH 9 with 5ꢀ
carboxytetramethylrhodamine succinimidyl ester (SEꢀTAMRA, 1.0 mg, Setareh Biotech)
in 100 % DMSO (Figure Sꢀ1). The reaction was incubated overnight at room temperature
protected from light to avoid photobleaching of the fluorophore. The resulting product
was purified by reverseꢀphase HPLC (Waters 2487) using a linear gradient consisting of
0.1 % trifluoroacetic acid and acetonitrile on an analytical C18 column. Elution of
AdoHcyꢀTAMRA and AdoHcy were monitored spectroscopically at the absorption
maximum of the fluorophore (545 nm) and the adenine moiety of AdoHcy (260 nm)
using a dual wavelength detector (Waters 2487 Dual λ Absorbance Detector). Eluted
compounds demonstrating absorption at both wavelengths were collected and further
characterized using ESIꢀMS (Finnigan LCꢀQꢀDeca) in positive ion mode.
Equilibrium Binding and Competitive FP Assays with AdoHcy-TAMRA
All FP assays were conducted using a Biotek Synergy H4 Hybrid Reader to measure the
parallel (F||) and perpendicular (F⊥) fluorescence intensities, which were then used to
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calculate the mP value (Equation 1) . Samples for performing the equilibrium binding
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or competitive FP experiments were incubated for 10 minutes at ambient temperature
prior to measuring the fluorescence intensities. Equilibrium binding experiments to
determine the Kd value for the HpMTANꢀD198N/AdoHcyꢀTAMRA complex were
performed using 5 nM of AdoHcyꢀTAMRA with varying concentrations of HpMTANꢀ
D198N in 50 mM HEPES at pH 7.5. The resulting data were fitted to a one site ꢀ total
binding equation in Prism 7 (GraphPad Software, San Diego, CA) where mPmax
represents the maximum fluorescent polarization signal, NS is the slope for nonspecific
binding of the fluorescent probe, and mPmin is the minimum fluorescent polarization
signal (Equation 2).
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Equation 1:
Equation 2:
The competitive FP assays were conducted using a concentration of AdoHcyꢀTAMRA at
the Kd value and a concentration of HpMTANꢀD198N that allows for 70 – 80 % enzymeꢀ
probe complex. Therefore, the competitive FP experiments involved using a
concentration of 11.3 nM for AdoHcyꢀTAMRA and 50 nM for HpMTANꢀD198N with
varying concentrations of ATP, ADP, AMP, AdoHcy, AdoMet, or MTA. Determination
of the Ki values of all adenosineꢀcontaining compounds were fitted to a one site – fit Ki
(equation 3) from Prism 7 (GraphPad Software, San Diego, CA).
Investigating the effects of background AdoMet was assessed by generating
multiple displacement curves of AdoHcy in the presence of various concentrations of
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AdoMet. Additionally, the reproducibility for the HpMTANꢀD198N/AdoHcyꢀTAMRA
competitive FP assay was determined by measuring 13 replicates of positive and negative
reactions, which were used to determine the Z′ value (Equation 4) 28.
Equation 3:
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Equation 4:
Kinetic Characterization of EgtD and Alkaline Phosphatase using HpMTAN-
D198N/AdoHcy-TAMRA Competitive FP assay
app
Determining the Km
of Lꢀhistidine for the purified EgtD using the developed
competitive FP assay was performed at 25 °C. The assay components consisted of 50 nM
HpMTANꢀD198N, 11.3 nM AdoHcyꢀTAMRA, and 5 nM EgtD in 25 mM TRIS at pH
8.0. A stock solution of Lꢀhistidine was serially diluted in the kinetic reactions to provide
a concentration range from 0.05 to 3.00 mM. The kinetic reactions were initiated by the
addition of 500 nM of AdoMet, and the rate of catalysis of EgtD was continuously
monitored by observing a decrease in the mP value. Conversion of the observed initial
velocities into [AdoHcy]/minute was calculated using a standard curve of AdoHcy in the
presence of 500 nM AdoMet (Figure Sꢀ2). The resulting data were fitted to the
app
MichaelisꢀMenten equation using Prism 7 for calculating Km of Lꢀhistidine for EgtD
(Equation 5).
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Equation 5:
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Kinetic characterization of ATP hydrolysis by Calf Intestinal Alkaline Phosphatase
(CIAP, New England Biolabs) was performed similarly to the kinetic analysis of EgtD.
Briefly, an assay mixture consisting of 50 nM HpMTANꢀD198N, 11.3 nM AdyHcyꢀ
TAMRA, and 0.25 U/mL of CIAP in 25 mM TRIS at pH 8.0 were initiated by adding
ATP at varying concentrations ranging from 0.1 to 20 ꢀM. Steadyꢀstate analysis of the
resulting data was fitted to a substrateꢀinhibition equation using Prism 7 (Equation 6).
Equation 6:
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Pilot Screen and Hit Validation of NIH Clinical Collection with EgtD
The pilot screen involved screening EgtD against the NIH Clinical Collection, which
contains 446 compounds that were dissolved in 100 % DMSO to provide a stock
concentration of 10 mM for each compound. Briefly, screening the NIH Clinical
Collection consisted of adding 48.5 µL of an assay mixture (51.5 nM HpMTANꢀD198N,
11.6 nM AdoHcyꢀTAMRA, 11.1 nM EgtD, 773 µM Lꢀhistidine, and 51.5 mM TRIS at
pH 8.0) to a 384ꢀblack well plate, followed by the addition 0.5 µL of each tested
compound (100 µM, and 1 %DMSO) to each well. The reactions were initiated with 1 µL
of AdoMet (500 nM) and the rate of all reactions for EgtD were monitored continuously.
Screening hits were validated assessing the inhibitor doseꢀresponse to EgtD by serially
diluting the compound of interest and performing the assay as previously described.
Additionally, the robustness and reproducibility of the developed competitive FP assay
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for monitoring the activity of EgtD was assessed by calculating the Z′ value (Equation 3)
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using 13 positive and negative replicates.
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RESULTS AND DISCUSSION
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Design and Synthesis of AdoHcy-TAMRA
Previously, synthesized AdoHcy and AdoMet analogues possessed a fluorophore on the
adenine or 5’ꢀthio moieties 23,29,30, but this was avoided in the design of the fluorescent
probe for HpMTANꢀD198N. The active site cavity of HpMTAN is compact, and
accommodation of a fluorophore conjugated to either of the aforementioned moieties
would potentially disrupt essential interactions between the fluorescent probe and
residues in the MTAN active site cavity. Conversely, examination of the crystal structure
for HpMTANꢀD198N complexed with AdoHcy illustrates that the αꢀamino moiety of
AdoHcy is not buried in the active site, but is positioned at the protein surface (Figure 2)
25. We envisioned that placement of a fluorophore on the αꢀamino moiety of AdoHcy
would maintain essential interactions for the fluorescently labeled ligand within the
active site cavity and promote highꢀaffinity binding of the fluorescent probe. The
synthetic approach for AdoHcyꢀTAMRA involved using nucleophilic acyl substitution in
a oneꢀpot synthesis by combining AdoHcy with SEꢀTAMRA, which is a rhodamine
derivative containing an amineꢀreactive moiety (Figure Sꢀ1). In slightly basic conditions,
the αꢀamino group of AdoHcy attacks the electrophilic center of the succinimidyl ester
linkage of SEꢀTAMRA, resulting in the formation of an amide linkage between TAMRA
and AdoHcy. The synthesized AdoHcyꢀTAMRA probe was separated from the reaction
mixture using reverse phase HPLC (Figure Sꢀ3), and the appropriate eluting peak was
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immediately validated by ESIꢀMS providing the expected mass of 797.69 Da (Figure Sꢀ
4).
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Figure 2: The surface model of the inactive variant HpMTAN-D198N complexed with AdoHcy (PDB:
4OY3) illustrating the rationale for modifying the α-amino moiety of AdoHcy with TAMRA. The
enzyme exists as a biological homodimer, which is represented in green and grey. The crystal
structure of HpMTAN-D198N complexed with AdoHcy highlights the α-amino moiety positioned at
the surface of the protein. Therefore, conjugation of a fluorophore to the α-amino moiety avoids
disrupting essential interactions for ligand binding within the compact active site cavity.
Assessing the Affinity of AdoHcy-TAMRA to HpMTAN-D198N
Determining the Kd value for AdoHcyꢀTAMRA to HpMTANꢀD198N was performed in
nonstoichiometric conditions by varying the concentration of the inactive variant while
maintaining the concentration of the fluorescent probe significantly below the previously
measured Km value of 5.0 ꢀM for AdoHcy 25. Intriguingly, the generated binding
isotherm demonstrates that AdoHcyꢀTAMRA possesses high affinity to HpMTANꢀ
D198N exhibiting a determined Kd value of 11.3 ± 0.7 nM (Figure 3). Previous kinetic
characterization of MTA and AdoHcy for HpMTAN has shown these substrates to
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possess low ꢀM affinity by measuring the spectral change of the reaction through
formation of adenine 25,31. More recently, steadyꢀstate kinetic analysis using a luciferaseꢀ
based assay for Staphylococcus aureus MTAN indicated negative cooperativity between
the two monomers demonstrating Km values for MTA of 100 nM and 900 nM for the first
and second active sight, respectively 32. The binding isotherm of AdoHcyꢀTAMRA
presented here represents the affinity measured for the first active site binding event,
which affords the high sensitivity of the developed FP assay. Fluorescent probes
possessing high affinity for their target exhibit several benefits including being cost
effective, reduced aggregation of fluorophores, and the capability of resolving a wide
range of ligand affinities. A common misconception is that competitive FP assays with
tightꢀbinding fluorescentlyꢀlabeled ligands should be avoided when measuring the
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potency of inhibitors with intermediate affinity . However, tightꢀbinding fluorescently
labeled ligands provide an extended range of inhibitor potency in comparison to moderate
affinity fluorescentlyꢀlabeled ligands. Assessment of the reproducibility of the
HpMTANꢀD198N/AdoHcyꢀTAMRA FP assay was determined by calculating the Z′
value using the concentrations of the fluorescent probe and enzyme implemented in the
competitive FP assay. The Z′ value is a statistical measure that evaluates the quality of
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the assay, where calculated values greater than 0.5 demonstrate a suitable assay . The
determined Z′ for the developed competitive FPꢀbased assay was determined to be 0.77,
validating the reproducibility and dynamic range of the assay.
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Figure 3: The binding isotherm of AdoHcy-TAMRA to HpMTAN-D198N. The resulting data are
presented in triplicates and were fitted to a one-site binding equation (Equation 2). The offset plot
represents the linear portion of the binding isotherm. The measured Kd value for AdoHcy-TAMRA to
HpMTAN-D198N was determined to be 11.3 ± 0.7 nM.
Quantification of AdoHcy and MTA from AdoMet-dependent enzymes
Adapting the HpMTANꢀD198N/AdoHcyꢀTAMRA complex for quantifying AdoHcy or
MTA generated from AdoMetꢀdependent enzymes is contingent on the sensitivity and
capability of differentiating the adenosineꢀbased products from substrates. For instance,
assays that monitor MTase activity require high sensitivity due to inherently slow
catalytic turnover or avoiding product inhibition at high concentrations of AdoHcy 13,20,33
.
We converted the developed HpMTANꢀD198N/AdoHcyꢀTAMRA complex into a
competitive FP assay that would directly detect AdoHcy or MTA by monitoring the
displacement of AdoHcyꢀTAMRA from HpMTANꢀD198N. The quality of the developed
competitive FP assay was determined by generating displacement curves for AdoHcy and
MTA to evaluate the affinity and limit of detection (LOD) of these adenosineꢀbased
molecules. Analysis of the displacement curves for MTA and AdoHcy demonstrate LOD
values measuring up to approximately 10 nM (0.5 pmol) and 6 nM (0.3 pmol) as well as
providing calculated Ki values of 24.2 ± 1.2 and 29.5 ± 3.6 nM (Figure 4), respectively.
Interestingly, the LOD of AdoHcy for the developed competitive FP assay is notably
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better or comparable to previously published spectroscopic, fluorescenceꢀbased or FP
immunoassays for measuring MTase activity 14,15,23,34. Several developed assays for
MTases require multiple coupled enzymes to manipulate generated products into
producing an output signal, which can lower the sensitivity of the assay. Coupling the
HpMTANꢀD198N/AdoHcyꢀTAMRA complex directly to AdoMetꢀdependent enzymes
provides a highly sensitive universal assay that immediately detects generated products
without the complication of additional auxiliary enzymes. A limitation with the
HpMTANꢀD198N/AdoHcyꢀTAMRA FP competitive assay is the narrow linear range for
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detecting AdoHcy in comparison to previously published AdoHcy quantificationꢀbased
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assays (Figure Sꢀ1)
.
Previously developed competitive FP immunoassays
demonstrate similar limitations in the linear ranges for detecting AdoHcy 23,34, which
presents a challenge for obtaining quantifiable kinetic parameters such as Km and kcat.
Figure 4: Displacement curves of MTA and AdoHcy using the HpMTAN-D198N/AdoHcy-TAMRA
competitive FP assay. Triplicates are directly plotted and the resulting data were fitted to equation 3.
The determined Ki values for MTA and AdoHcy are 24.2 ± 1.2 and 29.5 ± 3.6 nM, respectively.
Kinetic characterization of AdoMetꢀdependent enzymes typically requires relatively high
concentrations of AdoMet, potentially interfering with the HpMTANꢀD198N/AdoHcyꢀ
TAMRA competitive FP assay. Initially, we assessed the effect of background AdoMet
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displacing AdoHcyꢀTAMRA from HpMTANꢀD198N by generating a displacement curve
for the AdoHcy analogue. Analysis of the displacement curve for AdoMet presented an
unexpected Ki value of 989 ± 160 nM. Comparison of the displacement curves for the
adenosineꢀcontaining molecules illustrate that MTA and AdoHcy possesses a 34ꢀfold
higher affinity to HpMTANꢀD198N than AdoMet (Figure 5A). A challenge for coupling
the HpMTANꢀD198N/AdoHcyꢀTAMRA complex to monitor the activity of AdoMetꢀ
dependent enzymes is the observed moderate LOD for AdoMet, which was determined to
be 312 nM. Unfortunately, the effect of low ꢀM concentrations of AdoMet for
quantifying AdoHcy/MTA consequently reduces the signal window and slightly
increases the LOD of AdoHcy (Figure 5B). The highest AdoMet concentration that the
developed competitive FP assay tolerates was determined to be 5 ꢀM, which significantly
decreased the signal window to 70 ∆mP. Despite this interference of background AdoMet
at higher concentrations, the developed competitive FP assay clearly discriminates
between AdoHcy/MTA and AdoMet, resulting in a dynamic range that affords accurate
measurement of AdoHcy/MTA production in a workable background of AdoMet.
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Figure 5: Displacement curves of AdoMet and AdoHcy. Triplicates are demonstrated in all plots and
data were fitted to equation 3. (A) Generated displacement curves of AdoMet and AdoHcy by the
developed competitive FP assay highlighting the differentiation of the two adenosine-containing
molecules. The determined Ki values for AdoMet and AdoHcy were 989 ± 160 nM and 29.5 ± 3.6 nM,
respectively. (B) Displacement curves of AdoHcy in the presence of varying concentrations of
AdoMet. The effect of higher concentrations of AdoMet consequently increases the LOD of AdoHcy
and decreases the signal window for the developed competitive FP assay.
Kinetic Characterization and Pilot Screen of EgtD
Initially, the HpMTANꢀD198N/AdoHcyꢀTAMRA competitive FP assay was used to
examine the kinetic parameters of Lꢀhistidine methylation by an AdoMetꢀdependent
MTase enzyme, EgtD, encoded by Mtb using nonꢀsaturating steadyꢀstate kinetics. EgtD
catalyzes the initial step in the biosynthesis of ergothioneine by catalyzing the
trimethylation of the αꢀamino acid moiety of Lꢀhistidine (Figure 6A) 26,36. Previously,
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determination of the affinity for Lꢀhistidine and AdoMet to Mycobacterium smegmatis
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EgtD using isothermal calorimetry showed Kd values of 290 ± 14 and 270 ± 20 ꢀM ,
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respectively. Kinetic characterization of Lꢀhistidine by EgtD at nonꢀsaturating AdoMet
app
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concentrations using the developed competitive FP assay demonstrated a Km value of
330 ± 35 ꢀM (Figure 6B), which is within error of the previously determined Kd value for
the homolog. Pseudo firstꢀorder kinetics was not performed because of the interference of
background AdoMet at higher concentrations; however, steadyꢀstate kinetics can be
conducted to approximate the Km value for substrates using the developed competitive FP
assay. Once the kinetic parameters for EgtD were determined, the assay conditions were
optimized to maximize the dynamic range of the competitive FP assay to perform highꢀ
throughput screening. Validating the suitability of performing highꢀthroughput screening
with the optimized assay condition was evaluated by calculating the Z′ value, resulting in
an acceptable Z′ of 0.56.
Figure 6: (A) The enzymatic reaction for EgtD, which catalyzes the trimethylation of the α-amino
acid group of L-histidine. (B) The generated Michaelis-Menten curve for L-histidine to EgtD using
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the HpMTAN-D198N/AdoHcy-TAMRA competitive FP assay. The data were plotted as the mean
with the standard deviation of the three independent experiments. The determined Km for L-
histidine to EgtD was 330 ± 35 ꢀM.
app
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As a proof of concept, EgtD was screened against the NIH Clinical Collection to identify
potential inhibitors as well as demonstrate the capability of performing highꢀthroughput
screening with the developed competitive FP assay. Previously, EgtD has been shown to
be required for the biosynthesis of ergothioneine as well as being essential for the growth
of Mtb in murine macrophages and animal models 37ꢀ39. Analysis of the 446 compounds
screened from the NIH clinical collection yielded a hit cutꢀoff of 30.1 % activity of EgtD,
which produced an initial hit rate of 4.3 % (19 compounds, Figure 7A). The hit cutꢀoff
was determined by calculating 3 standard deviations from the average of the entire
screening data. Notably, the design of the developed competitive FP assay provides an
advantage when performing highꢀthroughput screening by easily identifying and reducing
the hit rate of interfering compounds with the assay. As stated previously, several current
available methods for MTases require multiple coupling enzymes, which complicates
highꢀthroughput screening by increasing the number of false positives. The developed
competitive FP assay simplifies highꢀthroughput screening for MTases by only requiring
the HpMTANꢀD198N/AdoHcyꢀTAMRA complex, and easily identifying compounds
interfering with the assay by observing an initial decrease in the signal window.
Inspection of the highꢀthroughput screening data demonstrated 3 compounds interfering
with the competitive FP assay by either significantly decreasing the signal window or
containing similar fluorescent properties as the fluorophore. The preliminary 19 hits from
the primary screen were validated by determining if the observed decrease in activity was
reproducible and if the small molecules exhibited a doseꢀresponse relationship to EgtD.
Out of the 19 hits, only 6 compounds reproduced a decrease in activity below the hit cutꢀ
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off as well as demonstrated a doseꢀresponse to EgtD (Figure 7B), concluding a final hit
rate of 1.4 % for screening of the NIH clinical collection. A potential challenge for
screening AdoMetꢀdependent MTases containing a Km value greater than 2.5 ꢀM of
AdoMet will be experiencing an initial increase in hit rates from identifying compounds
that bind weakly to the enzyme of interest. Indeed, it is commonly understood that the
choice of concentrations for substrates relative to an enzyme’s inherent Km values can
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alter screening results . The presented screening results for EgtD initially identified 13
false positives, which we attributed to performing the assay with an AdoMet
concentration significantly below the previously determined Kd value. Despite the
limitations of interfering background AdoMet at µM concentrations, the developed
competitive FP assay identified and validated 6 inhibitors from the NIH Clinical
Collection that demonstrate moderate affinity for EgtD.
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Figure 7: (A) Graphical representation of EgtD screened with the NIH clinical collection.
The plotted solid line represents the average of the entire screening data. Whereas, the
plotted dashed line represents the hit cutꢀoff, which was determined by taking the 3
standard deviation units from the average. Any compound that falls below the hit cutꢀoff
represents a preliminary hit. Initially, 19 compounds were identified, but after validating
only 6 compounds were confirmed as hits. These 6 compounds are circled on the
presented plot. (B) The doseꢀdependence assessment of the 6 confirmed hits form the
highꢀthroughput screen of EgtD with the NIH clinical collection.
Further Applications of the HpMTAN-D198N/AdoHcy-TAMRA Competitive FP Assay
We further explored applications of the HpMTANꢀD198N/AdoHcyꢀTAMRA
competitive FP assay for quantifying additional adenosineꢀbased molecules to expand the
versatility of the developed assay. In addition to catalyzing AdoHcy and MTA
hydrolysis, it has been shown that MTAN hydrolyzes the Nꢀribosidic bond for a variety
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of adenosineꢀcontaining molecules with various substituents on the 5’ position
.
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Intriguingly, it was serendipitously observed that the HpMTANꢀD198N/AdoHcyꢀ
TAMRA competitive FP assay detects AMP and ADP at moderate concentrations, while
ATP is unable to displace AdoHcyꢀTAMRA (Figure 8A). The capability of
differentiating AMP and ADP from ATP suggests that the developed competitive FP
method is an alternative assay for measuring the activity of ATPꢀdependent enzymes.
Analysis of the displacement curves for AMP and ADP provide measured Ki values of
933 ± 66 and 15.2 ± 1.3 ꢀM, respectively. The determined affinities of the adenosineꢀ
containing molecules illustrate that the developed competitive FP assay is more efficient
at monitoring enzymes that produce ADP. Currently, several assays exist commercially
to monitor the activity of kinases 42. Most of these assays do not tolerate a high
background concentration of ATP 43, whereas the developed competitive FP assay
tolerates up to 100 mM ATP. Our developed competitive FP assay allows for more than
one order of magnitude higher ATP concentration to be tolerated in comparison to
standard kinase assays. The advantages of tolerating a wide range of ATP concentrations
is that it can easily determine and accommodate a diverse range of Km values for ATP
exhibited by kinases as well as allow for ATP competitiveness studies 43.
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Figure 8: (A) Generated displacement curves of ATP, ADP and AMP using the HpMTAN-
D198N/AdoHcy-TAMRA competitive FP assay. Triplicates are demonstrated in all plots and data
were fitted to equation 3. The determined Ki values for ADP and AMP were 15.2 ± 1.3 and 933 ± 67
ꢀM, respectively. (B) Michaelis-Menten plot of alkaline phosphatase exhibiting the known substrate
inhibition curve. The resulting data were fitted to equation 6 resulting in Km values of 0.9 ± 0.2 ꢀM
and Ki value of 7.6 ± 1.8 ꢀM for ATP, respectively.
As a proof of concept, we examined the kinetic parameters of ATP hydrolysis by CIAP
using the developed HpMTANꢀD198N/AdoHcyꢀTAMRA competitive FP assay.
Previously, MichaelisꢀMenten kinetics performed on CIAP using a molybdenum blue
based assay exhibited substrate inhibition of ATP at low ꢀM concentrations, which
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resulted in a Km value of 20.5 ꢀM and a Ki value of 19.0 ꢀM . Steadyꢀstate analysis of
CIAP using the developed competitive FP assay demonstrates a Km value of 0.9 ± 0.2 ꢀM
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and Ki value of 7.6 ± 1.8 ꢀM for substrate inhibition of ATP (Figure 8B). The 8ꢀfold
difference in the determined Ki in comparison to the previously published value we
attribute to the assay reaction not containing an equivalent concentration of magnesium
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chloride, which is known to contribute to substrate inhibition of ATP . The lower Km
and Ki values measured for ATP is likely a result of the developed competitive FP assay
containing higher sensitivity in comparison to the molybdenum blue based assay. The
previous determined LOD for measuring free inorganic phosphate using the molybdenum
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blue based assay was determined to be 25 ꢀM . Additionally, it is well known that
CIAP sequentially hydrolyzes the inorganic phosphate groups from ATP 46, thereby
creating a mixture of analytes with differing affinities to MTAN. At any point in time, the
results exhibit an overall increase in adenosine nucleotides and adenosine, but, in this
specific application, the developed competitive FP assay cannot distinguish between
ADP, AMP, or adenosine. The LOD demonstrated by the developed competitive FP
assay for ADP was determined to be 0.5 ꢀM, providing a 50ꢀfold increase in sensitivity
in comparison to the molybdenum blue based assay. Alternatively, adenosine is
structurally analogous to MTA and 5′ꢀdeoxyadenosine, which are hydrolyzed by MTAN
to avoid product inhibition of polyamine biosynthesis and AdoMet radical pathways,
respectfully 47. Since the developed competitive FP assay demonstrates a LOD of 10 nM
for MTA, it is likely that adenosine possesses similar affinity to HpMTANꢀD198N and
would provide a 2500ꢀfold increase in sensitivity in comparison to the molybdenum blue
assay. Ultimately, most applications for this assay will be toward enzymes with a known
single adenosineꢀcontaining product, and the observed lack of discrimination specific to
the coupled alkaline phosphatase assay would not be relevant in the other applications.
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CONCLUSION
The work presented describes an alternative method for universally detecting generated
AdoHcy from MTases that is highly sensitive, amenable for highꢀthroughput screening
and performed in a simple mixꢀandꢀread format. Besides monitoring the activity of
MTases, our developed competitive FP assay is applicable to several AdoMetꢀdependent
and ATPꢀdependent enzymes because of the promiscuity of MTAN binding various
adenosineꢀcontaining molecules. Additionally, our developed FP assay allows for a
feasible method for highꢀthroughput screening as well as determination of binding
constants for ligands to the various homologs of MTAN.
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ACKNOWLEDGMENTS
A portion of this research was also supported by the National Institute of Allergy and
Infectious Disease (NIAID)/National Institutes of Health (NIH) to D.R.R (AI105084).
SUPPORTING INFORMATION
Cloning and purification of HpMTANꢀD198N and EgtD, oneꢀpot synthetic scheme for
AdoHcyꢀTAMRA (Figure Sꢀ1), standard curve of AdoHcy with 500 nM AdoMet (Figure
Sꢀ2), HPLC chromatogram for the purification of AdoHcyꢀTAMRA (Figure Sꢀ3), and the
mass spectrum of purified AdoHcyꢀTAMRA (Figure Sꢀ4).
Supplemental.docx
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