Analytical Biochemistry 544 (2018) 80–86

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Analytical Biochemistry
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A continuous assay for L-talarate/galactarate dehydratase using circular T

dichroism
Nicole M. Eastona,1, Sarah A.E. Aboushawareba,1, Stephen L. Bearnea,b,∗
a
Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada
b
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: L-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the
Dehydratase dehydration of either meso-galactarate or L-talarate to form 5-keto-4-deoxy-D-glucarate (5-KDG). To facilitate
Assay study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been
Circular dichroism developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed
Kinetics
conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The
m-Galactarate
apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ± 2 deg cm2 dmol−1, which
5-Keto-4-deoxy-D-glucarate
was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters
Km, kcat, and kcat/Km were 0.38 ± 0.05 mM, 4.8 ± 0.1 s−1, and 1.3 ( ± 0.2) × 104 M−1s−1, respectively. These
values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry 46,
9564–9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant
(Ki = 10.7 ± 0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay
offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase,
and to the labor-intensive, fixed-time assays.

Introduction
dehydratase (EC 4.2.1.42, TGD1) is a member
L-Talarate/galactarate
of the mandelate racemase (MR) subgroup of the enolase superfamily.
Members of the MR subgroup share with other members of the enolase
superfamily a common bidomain structure (i.e., (β/α)7β-barrel domain
and an α+β capping domain) and a common partial reaction (i.e., the
metal-assisted, Brønsted base-catalyzed abstraction of the α-proton
from a carboxylate substrate to form an enol(ate) intermediate) [1–4].
TGD catalyzes the dehydration of either meso-galactarate or L-talarate
to form 5-keto-4-deoxy-D-glucarate (5-KDG) as illustrated in Scheme 1
[5]. Despite the fact that the MR subgroup is named for its archetype
MR, most enzymes in the subgroup, such as TGD, catalyze a dehydra-
tion reaction of a sugar acid substrate rather than a racemization re-
action [6,7]. Although the catalytic machinery responsible for over-
coming the kinetic and thermodynamic barriers accompanying
deprotonation of a weak carbon acid substrate and the overall struc-
tural fold have been conserved within the MR subgroup, divergent
evolution has led to a variety of dehydratases that act on specific
aldonic and aldaric acid substrates. These features of the subgroup, and
indeed the superfamily, have made annotation of function difficult
[8–10] and numerous detailed studies to understand the structure-
function relationships among the members of the MR subgroup have
focused on delineating the substrate specificity of the enzymes
[5–7,11–20]. In addition to delineating the biochemical roles of dehy-
dratases in carbohydrate metabolism, these enzymes have received at-
tention in recent years because the dehydratase-catalyzed formation of
2-ketoaldaric acids affords a route to deoxygenating carbohydrates in
the biomass for use as biofuels, and to producing precursors for the
synthesis of value-added chemicals [21,22]. Such studies require con-
venient assays for the enzymes, preferably ones that are direct and
continuous. Several assays have been developed for galactarate dehy-
dratases, including a coupled assay that utilizes 5-keto-4-deoxy-D-glu-
carate aldolase and L-lactate dehydrogenase as the coupling enzymes
[5], and fixed-time assays that involve detection of the product through
derivatization with semicarbazide [12,16,23] or treatment with peri-
odate and thiobarbituric acid [24]. In addition, the enzyme-catalyzed
hydrogen-deuterium exchange at the α-position of the substrate has

Abbreviations: CD, circular dichroism; (His)6, N-terminal hexahistidine tag; 5-KDG, 5-keto-4-deoxy-D-glucarate; MR, mandelate racemase; TGD, L-talarate/galactarate dehydratase;
StTGD, TGD from Salmonella typhimurium; Tris, tris(hydroxymethyl)aminomethane
∗
Corresponding author. Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada.
E-mail address: sbearne@dal.ca (S.L. Bearne).
1
Both authors contributed equally to the work.

https://doi.org/10.1016/j.ab.2017.12.015
Received 29 August 2017; Received in revised form 30 November 2017; Accepted 11 December 2017
Available online 14 December 2017
0003-2697/ © 2017 Elsevier Inc. All rights reserved.
N.M. Easton et al.
Scheme 1. StTGD-catalyzed dehydration of m-galactarate and L-talarate to yield 5-KDG.
been used to follow the reaction by 1H NMR spectroscopy [5,12] and
polarimetry has been used to follow the change in optical rotation
[5,12,16]. Some of these assay methods have their own particular
disadvantages. For example, the coupled assay is limited by the fact that
the aldolase is not readily available, specific conditions must be met for
optimal functioning of the coupling enzymes, and the assay may not be
amenable to inhibition studies because of the potential for structural
similarity of inhibitors to the substrate for the coupling enzyme. Neither
the fixed-time assays nor the 1H NMR-based assay permit rapid de-
termination of initial rates, and the latter may have a solvent kinetic
isotope effect when the reaction is conducted in D2O. However, re-
cognition that m-galactarate has the meso configuration and is optically
inactive, while the product 5-KDG is optically active (αD25 = +45°)
[5], led us to develop a circular dichroism (CD)-based assay for TGD
activity. This method is related to the polarimetric assay and could be
beneficial if a polarimeter is not available. Using the TGD from Sal-
monella typhimurium (StTGD) and m-galactarate as the substrate, we
show that the CD-based assay offers a quick, practical, and effective
method for monitoring TGD activity.
Materials and methods
General
Tartronic acid was obtained from Alfa Aesar (Tweksbury, MA).
Mucic acid (m-galactaric acid) and all other chemicals were purchased
from Sigma-Aldrich Canada Ltd. (Oakville, ON). Synthetic deoxy-oli-
gonucleotide primers were purchased from Integrated DNA
Technologies (Coralville, IA). Restriction endonucleases were pur-
chased from New England Biolabs (Ipswich, MA). An S1000 Thermal
Cycler (Bio-Rad Laboratories, Mississauga, ON) was used for poly-
merase chain reactions (PCR). A Branson Sonifier 250 was used for
sonication. His·Bind resin was purchased from Novagen (Madison, WI).
All other chemicals were reagent grade or better. Assays were con-
ducted using a Jasco J-810 spectropolarimeter (Jasco, Inc., Easton,
MD). 1H NMR analyses were conducted using a Bruker AV-500 spec-
trometer at the Nuclear Magnetic Resonance Research Resource (NMR3,
Dalhousie University).
Cloning
The open reading frame encoding StTGD (GI: 16766982) was cloned
from genomic DNA of Salmonella typhimurium LT2 using Phusion High-
Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). The
PCR-based amplification reaction was conducted as reported by Gerlt
and co-workers [5] using the forward (5′-GTGATTATCAGGAGAAAAC
ATATGGCTTTAAGCGCGAATTCCG-3′) and reverse (5′-GATTCCCGCCA
GGATCCTTAAGGGCGTTTGCCAAATTCAC-3′) primers indicated, where
the underlined bases correspond to NdeI and BamHI recognition sites,
81
Analytical Biochemistry 544 (2018) 80–86
respectively. The amplification parameters were adapted from those
reported by Gerlt and co-workers [5] as follows: initial denaturation at
98 °C for 30 s, 40 cycles of 94 °C for 60 s for denaturation, thermal
gradient of 45–60 °C for 75 s for annealing, 68 °C for 120 s for extension,
and 72 °C for 144 s for final extension. The amplification product,
purified using a QIAquick Gel Extraction Kit (Qiagen, Toronto, ON),
and the pET-15b vector were then digested separately in a stepwise
manner with NdeI for 2 h followed by BamHI for 2 h at 37 °C, according
to the manufacturer's instructions. Subsequently, the digestion products
were purified as mentioned above and ligation with T4 DNA ligase
(Invitrogen/Thermo Fisher, Waltham, MA) was conducted overnight at
16 °C following the manufacturer's instructions.
The pET-15b-StTGD plasmid encodes a fusion protein bearing an N-
terminal His6-tag. Chemically-competent E. coli DH5α cells were
transformed with the plasmid using heat shock, and glycerol stocks
were prepared and stored at −80 °C using standard protocols [25]. The
sequence of the insert was verified by commercial DNA sequencing
(Robarts Research Institute, London, ON) to ensure incorporation of the
insert and the absence of any mutations. Competent E. coli BL21 (DE3)
cells were also transformed with the pET-15b-StTGD plasmid to be used
for protein expression, and glycerol stocks were prepared and stored at
−80 °C.
Enzyme purification
StTGD was overexpressed and purified using a protocol similar to
that described in the Novagen manual [26]. Four starter cultures, each
containing sterile Luria-Bertani (LB) medium (5 mL), ampicillin (25 μg/
mL), and the glycerol stock (10 μL), were incubated overnight at 37 °C
with continuous shaking at 250 rpm. These starter cultures were then
added to sterile LB medium (10 mL per 1 L) containing ampicillin
(25 μg/mL), which was then incubated for 2 h at 37 °C followed by
24 h at 27 °C (without induction by isopropyl β-D-1-thiogalactopyrano-
side (IPTG) [5]) with continuous shaking at 250 rpm. The cells were
harvested using centrifugation (3000 × g, 10 min, 4 °C). The cell pellet
was re-suspended in 30 mL of ice-cold binding buffer [Tris-HCl buffer
(20 mM, pH 7.9) containing NaCl (0.5 M) and imidazole (5 mM)]. Fol-
lowing sonication on ice (5 × 30 s bursts with cooling for 1 min be-
tween bursts), the cell lysate was then clarified by ultracentrifugation
(110,000 × g, 30 min, 4 °C), and then passed through a Ni2+-charged
His·bind resin-packed column (10-mL columns packed to 2.5 mL) at
4 °C. The column was subsequently washed with binding buffer
(25 mL), wash buffer [15 mL, Tris-HCl (20 mM, pH 7.9) containing NaCl
(0.5 M) and imidazole (60 mM)], and strip buffer [7 mL, Tris-HCl
(20 mM, pH 7.9) containing NaCl (0.5 M) and EDTA (100 mM)]. The
majority of the protein eluted in the strip buffer and the purity was
assessed using 10% acrylamide SDS-PAGE followed by staining with
Coomassie blue R-250. StTGD was then dialyzed against assay buffer
[Tris-HCl (50 mM, pH 8.0) containing MgCl2 (10 mM)] for 24 h at 4 °C
N.M. Easton et al.
[5]. The enzyme preparation was subsequently diluted 2-fold by addi-
tion of assay buffer and stored at −20 °C. Enzyme concentrations were
determined using either the Bio-Rad protein assay (Bio-Rad Labora-
tories, Mississauga, ON, Canada) with BSA standards, or by measuring
the absorbance at 280 nm using the extinction coefficient for the re-
combinant protein of 59,930 M−1cm−1 calculated using the ExPASY
ProtParam tool [27]. The N-terminal His6-tag was not removed.
Determination of assay wavelength
To determine the appropriate wavelength for following the con-
version of m-galactarate to 5-KDG, CD spectra (wavelength range:
250–400 nm) were obtained for the assay buffer, m-galactarate (6.0 mM
in assay buffer), StTGD (25 ng/μL in assay buffer), and then for a
mixture of TGD with m-galactarate for 5 min-intervals up to 60 min
while incubating the reaction at 25 °C. A quartz cuvette with a 0.5-cm
light-path was used and the final volume of the reaction was 1.0 mL.
Molar ellipticity
To convert the observed changes in ellipticity to changes in con-
centration, the apparent molar ellipticity of 5-KDG was determined. m-
Galactarate (2.0–6.0 mM) was incubated at 25 °C with StTGD (50 ng/
μL) in Tris-HCl buffer (50 mM, pH 8.0) containing MgCl2 (10 mM) and
the reaction was followed at 323 nm in quartz cuvette with a 0.5-cm
light-path until the reaction was complete. Product analysis was con-
ducted by incubating a reaction mixture (25-mL total volume) that
contained m-galactarate (initial concentration of 6.0 mM), StTGD
(50 μg/mL), and MgCl2 (10.0 mM) in Tris-HCl buffer (50 mM, pH 8.0) at
37 °C until the reaction was complete as judged by the change in el-
lipticity at 323 nm. The protein was then removed by centrifugal fil-
tration using an Amicon Ultra-15 filtration device with a 10-kDa MWCO
that had been pre-washed with H2O and NaOH (0.1 M). The pH was
then adjusted to ∼5 using HCl and the sample was applied to a column
(1.4 × 15 cm) containing Dowex-50-X8 (Na+-form) to remove the Tris
buffer. Fractions containing the product were pooled and the solvent
removed by lyophilization. The resulting powder was dissolved in H2O
containing 10% D2O and analyzed using 1H and 13C NMR spectroscopy
and mass spectrometry.
TGD assay
Stock solutions of the substrate were prepared by first dissolving
both m-galactarate and Tris base in water, followed by dropwise addi-
tion of a solution of MgCl2, and then adjusting the pH to 8.0 using HCl.
The conversion of m-galactarate to 5-KDG was followed by measuring
the change in ellipticity at 323 nm for 3–5 min at 25 °C. To determine
the concentrations of StTGD that gave linear time courses, the assay was
conducted using m-galactarate concentrations of 0.1 mM and 6.0 mM
with varying enzyme concentrations (2.0–30.0 ng/μL). For routine as-
says, reaction mixtures contained StTGD (25 ng/μL), m-galactarate
(0.1–6.0 mM), Tris-HCl buffer (50 mM, pH 8.0), and MgCl2 (10 mM) in
a total volume of 1.0 mL. Nonlinear regression analysis was used to fit
equation (1) to initial rate data to obtain the kinetic parameters. Assays
were conducted in triplicate and the reported values are the averages
and the error is the standard deviation.
Vmax [S]
vi =
Km + [S]
Inhibition studies
Initial velocities were measured as described above. Reactions
contained m-galactarate (0.25, 0.50, 1.0, 2.5, and 5.0 mM) and
Analytical Biochemistry 544 (2018) 80–86
tartronic acid (0.0, 8.0, 16.0, and 24.0 mM; pH adjusted to 8.0) in Tris-
HCl buffer (50 mM, pH 8.0) containing MgCl2 (10 mM). The reaction
was initiated by addition of StTGD (final concentration of 25 ng/μL).
Nonlinear regression analysis was used to fit equation (2) to initial rate
data to obtain the kinetic parameters followed by re-plotting of the
apparent Km/Vmax values against the inhibitor concentration to esti-
mate the value of Ki in accord with equation (2). Inhibition assays were
conducted in triplicate and the reported Ki value is the average and the
error is the standard deviation.
Vmax [S]
vi =
(
Km 1 +
[I]
Ki ) + [S] (2)
Results and discussion
Direct continuous enzyme assays offer numerous advantages be-
cause they permit rapid determination of initial rates and do not limit
assay conditions to those that might be dictated by the other enzymes
employed in a coupled assay system. Consequently, we sought to de-
velop a direct continuous assay for TGD to facilitate studies on this
member of the enolase superfamily, as well as on other galactarate
dehydratases. The open reading frame encoding the enzyme from
Salmonella typhimurium was cloned into a pET-15b vector as reported
previously by Gerlt and co-workers [5]. The enzyme, bearing an N-
terminal His6-tag, was purified using metal ion affinity chromato-
graphy. We noted that the His6-tagged enzyme was quite prone to
precipitation on the His·Bind resin, necessitating occasional removal of
the precipitate during the addition of cell lysate to the column. Al-
though a substantial amount of the desired protein was lost in the
precipitate (Fig. 1, lane 2), the yield (∼7 mg/L culture) of pure protein
(Fig. 1, lane 8) was satisfactory for subsequent enzymatic studies. The
precipitation problem was not ameliorated by the inclusion of MgCl2
(5 mM) in the buffer [5], so magnesium was not included in the buffers
used during the purification, except for the final dialysis buffer. Finally,
we note that the enzyme was only stable for about 2–3 weeks at −20 °C.
For a CD-based assay to be feasible, the substrate and product
should exhibit substantially different ellipticities at the assay wave-
length so that the change in ellipticities can be observed during the
course of the enzyme-catalyzed reaction. Since m-galactarate is opti-
cally inactive, we surmised that the optically active 5-KDG should af-
ford a CD signal that could be used to follow the conversion of m-ga-
lactarate to 5-KDG. Although StTGD catalyzes a competing
epimerization reaction between the two substrates m-galactarate and L-
talarate along with the dehydration reaction, m-galactarate was chosen
(1)
Fig. 1. SDS-PAGE gel (10%) showing the purification of StTGD. Lane 1, molecular weight
marker; lane 2, precipitate; lane 3, crude cell lysate; lane 4, flow-through; lane 5, binding
buffer eluant; lane 6, wash buffer eluant; lane 7; strip buffer eluant; and lane 8, purified
StTGD post-dialysis. The calculated MW of StTGD based on the amino acid sequence is
46,564 Da.
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N.M. Easton et al. Analytical Biochemistry 544 (2018) 80–86

Fig. 2. CD spectral changes during the StTGD-catalyzed conversion of m-galactarate to 5-

KDG at 25 °C. CD spectra were recorded between 250 and 400 nm for the assay reaction
mixture containing m-galactarate (6.0 mM) and StTGD (25 ng/μL) in Tris-HCl buffer
(50 mM, pH 8.0) containing MgCl2 (10 mM) at various time periods up to 60 min. The
final volume of the reaction was 1.0 mL and the light path was 0.5 cm. The spectra of the
buffer, m-galactarate (6.0 mM in buffer), and StTGD (25 ng/μL in buffer) did not differ
from the zero time spectrum shown in the figure.

as the substrate because Gerlt and co-workers had previously reported

that 30% of L-talarate undergoes epimerization to form m-galactarate,
whereas epimerization of m-galactarate to L-talarate is negligible [5]. In
addition, m-galactarate (mucic acid) is commercially available.

Molar ellipticity determination

To determine the appropriate wavelength for following the reaction,

CD spectra (wavelength range: 250–400 nm) for the assay buffer, m-
galactarate, StTGD, and the reaction mixture of TGD with m-galactarate
at various times were obtained (Fig. 2). As m-galactarate was con-
sumed, the growth of a signal in the CD spectrum with a maximum
ellipticity at 323 nm was observed. Since m-galactarate exhibited neg-
ligible ellipticity at this wavelength, 323 nm was chosen as the optimal
wavelength at which to follow the reaction. Analysis of the StTGD- Fig. 3. Determination of the apparent molar ellipticity for 5-KDG. Progress curves (A) are
catalyzed reaction product using 1H and 13C NMR spectroscopy de- shown for complete StTGD-catalyzed (50 ng/μL) conversion of various initial con-
monstrated complete conversion to 5-KDG with the resulting 1H and centrations of m-galactarate (2.0 mM, ◯; 3.0 mM, △; 4.0 mM, ▽; 5.0 mM, □; and
13
C NMR spectra matching that of the published spectrum of 5-KDG 6.0 mM, ◇) to 5-KDG. The final changes in ellipticity were plotted as a function of the
initial concentration of m-galactarate (B) and the molar ellipiticity ([θ]) determined from
(see Supporting Information Fig. S1) [28]. In addition, low- and high-
the slope of the line. The experiment was conducted in triplicate and a representative trial
resolution mass spectrometry also confirmed dehydration of the sub-
is shown. The average slope was 1.01 ± 0.01 mdeg/mM (l = 0.5 cm), which gave
strate (see Supporting Information Figs. S2 and S3). By following the [θ]323 = 202 ± 2 deg cm2 dmol−1.
complete conversion of various initial concentrations of m-galactarate
to 5-KDG (Fig. 3A), the apparent molar ellipticity of 5-KDG at 323 nm
Δc (Δθ/Δt )
([θ]323) was determined to be 202 ( ± 2) deg cm2 dmol−1, using vi {in mM/s} = = = 0.99(Δθ/Δt ){in mdeg/s}
Δt [θ] l (4)
equation (3), where [θ] is the molar ellipticity (deg cm2 dmol−1), θ is
the observed ellipticity (deg), c is the concentration (M), and l is the
light-path length (cm).
Assay development
θ= [θ] lc (3)
Linear progress curves were obtained over the first 5 min and the
The molar ellipticity could then be used to convert observed initial values of Δθ/Δt were used to calculate the initial velocities as outlined
rates (i.e., Δθ/Δt in units of mdeg/s) into concentration-dependent above (data not shown). The validity of the continuous assay was es-
terms using equation (4), where l = 0.5 cm. tablished by demonstrating that the initial velocities for conversion of

83
N.M. Easton et al.
Fig. 4. Effect of enzyme concentration on the rate of the StTGD-catalyzed conversion of
m-galactarate to 5-KDG (A) and Michaelis-Menten plot (B). Panel A: The concentrations of
m-galactarate were 0.1 mM (▾) and 6.0 mM (▴). Other conditions are as described in the
Materials and methods. The initial velocities show a linear dependence on enzyme con-
centration between 2.0 and 30 ng/μL. Therefore, the concentration of StTGD chosen
within this range for standard kinetic analyses was 25 ng/μL. Panel B: Representative
Michaelis-Menten plot for the StTGD-catalyzed conversion of m-galactarate to 5-KDG. The
values of Km and Vmax are 0.38 ± 0.05 mM and 2.56 ± 0.05 μM/s, respectively, and the
corresponding kinetic constants are given in Table 1. The concentration of StTGD was
25 ng/μL and the experimental conditions were as described in the Materials and
methods.
m-galactarate to 5-KDG varied linearly with StTGD concentrations
(2.0–30.0 ng/μL) at both low (0.1 mM) and high (6.0 mM) substrate
concentrations (Fig. 4A). Consequently, 25 ng/μL was chosen as the
standard concentration of StTGD to be employed in the assay.
One disadvantage of CD-based assays is that they are limited by the
total absorbance of the sample, especially when the assay wavelength
is < 250 nm since the increase in the total absorbance of the reaction
mixture arising from elevated concentrations of buffer components and
ligands can substantially decrease the signal-to-noise ratio. For ex-
ample, CD-based assays of amino acid racemases are often
84
Analytical Biochemistry 544 (2018) 80–86
Table 1
Kinetic parameters for the StTGD-catalyzed conversion of m-galactarate to 5-KDG.
Source Km (mM) kcat (s−1) kcat/Km (M−1s−1)b
This studya 0.38 ± 0.05 4.8 ± 0.1 1.3 ( ± 0.2) × 104
Ref. [5] 0.33 ± 0.07 3.6 ± 0.2 1.1 × 104
a
Assays were conducted in triplicate with the concentration of StTGD equal to 25 ng/μL.
Errors are standard deviations.
b
The kcat/Km value and associated error was calculated from the values of kcat and Km
determined individually by fitting of equation (1) to the initial velocity data.
compromised by a requirement for substrate concentrations in the high
mM range due to their typically high Km values or by high concentra-
tions of inhibitors that exhibit weak binding affinities [29,30]. The
present assay offers a particular advantage in this regard since the
optimal assay wavelength is 323 nm, well above the absorbance of most
buffer components and small molecule ligands.
Kinetic parameters
Kinetic studies were conducted using the His6-tagged StTGD (Fig. 4)
and the kinetic parameters were determined by fitting equation (1) to
the initial velocity data (Table 1). The values of the kinetic parameters
are in excellent agreement with those obtained by Gerlt and co-workers
[5]. These authors used StTGD from which the N-terminal His6-tag had
been cleaved, while the present study was conducted with His6-tagged
enzyme, demonstrating that the tag has a negligible effect on the ob-
served kinetic parameters of StTGD.
Inhibition studies
To demonstrate the utility of this assay, we conducted an inhibition
study with tartronate. Previously, tartronate had been shown to be a
good competitive inhibitor of MR, binding with an inhibition constant
of 1.8 mM [31]. Interestingly, the X-ray crystal structure of the MR-
tartronate complex revealed that the glycolate moiety of tartronate
chelated the Mg2+ and that the distal carboxylate bridged the side
chains of the two active site Brønsted acid-base catalysts Lys 166 and
His 297. Since the active site architecture of MR is similar to that of
StTGD, which also contains Lys and His residues with similar orienta-
tions [5], we anticipated that tartronate should also inhibit StTGD.
Indeed, as the representative Lineweaver-Burk plot (Fig. 5) demon-
strates, tartronate is a competitive inhibitor of StTGD with a Ki value of
10.7 ± 0.4 mM. Interestingly, it is a much weaker inhibitor of StTGD
(Ki/Km ≈ 28) than MR (Ki/Km ≈ 2) [31]. This difference may arise
from subtle differences in either the precise location or the prontona-
tion states of the active site Brønsted acid-base catalysts that facilitate a
dehydration reaction in the case of StTGD versus a racemization reac-
tion in the case of MR.
In summary, we have developed a convenient, direct continuous
CD-based assay for following the TGD-catalyzed conversion of m-ga-
lactarate to 5-KDG. The advantages that this assay method offers over
the existing assays are that initial rate measurements may be obtained
much more rapidly than is possible using fixed-time assays (e.g.,
semicarbazide derivatization or NMR methods) and it is much less
complex than a coupled assay, which is often limited by the availability
and specific properties of the coupling enzyme(s). Furthermore, the
utility of the assay has been demonstrated through determination of the
inhibition constant for the competitive inhibitor tartronate. In addition
to being extremely useful for studying the enzymology of StTGD, this
assay should also be generally applicable to the assay of other ga-
lactarate dehydratases.
N.M. Easton et al.
Fig. 5. Inhibition of StTGD by tartronate. Panel A: Representative Lineweaver-Burk plot
demonstrating competitive inhibition of StTGD activity by tartronate. The concentrations
of m-galactarate ranged between 0.25 and 5.00 mM and the concentrations of tartronate
were 0.0 mM (●), 8.0 mM (▴), 16.0 mM (▾), and 24.0 mM (▪). Other conditions are as
described in the Materials and methods. Panel B: Replot of the apparent Km/Vmax values
obtained using nonlinear regression analysis as a function of tartronate concentration.
The Ki value is 10.7 ± 0.4 mM.
Acknowledgment
We thank the Natural Sciences and Engineering Research Council
(NSERC) of Canada for a Discovery Grant (S.L.B.; RGPIN-2016-05083)
and an Undergraduate Summer Research Award (N.M.E.). N.M.E. and
S.A.E.A. were recipients of a Faye Sobey Undergraduate Research
Award and a Nova Scotia Graduate Scholarship, respectively. We also
thank Dr. John Rohde (Dalhousie University) for kindly providing
genomic DNA from Salmonella typhimurium, and Dr. Mike Lumsden
(NMR3, Dalhousie University) for his assistance with NMR experiments.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.ab.2017.12.015.
85
Analytical Biochemistry 544 (2018) 80–86
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