Botting School of Chemistry, University of St Andrews, St Andrews, Fife, Scotland, UK Recombinant human kynureninase L-kynurenine hydrol-ase, EC 3.7.1.3 was purified to homogeneity 60-fol
Trang 1Purification and biochemical characterization of some
of the properties of recombinant human kynureninase
Harold A Walsh and Nigel P Botting
School of Chemistry, University of St Andrews, St Andrews, Fife, Scotland, UK
Recombinant human kynureninase (L-kynurenine
hydrol-ase, EC 3.7.1.3) was purified to homogeneity (60-fold) from
Spodoptera frugiperda(Sf9) cells infected with baculovirus
containing the kynureninase gene The purification protocol
comprised ammonium sulfate precipitation and several
chromatographic steps, including DEAE–Sepharose
CL-6B, hydroxyapatite, strong anionic and cationic
sepa-rations The purity of the enzyme was determined by SDS/
PAGE, and the molecular mass verified by MALDI-TOF
MS The monomeric molecular mass of 52.4 kDa
deter-mined was > 99.99% of the predicted molecular mass A
UV absorption spectrum of the holoenzyme resulted in a
peak at 432 nm The optimum pH was 8.25 and the enzyme
displayed a strong dependence on the ionic strength of the buffer for optimum activity This cloned enzyme was highly specific for 3-hydroxykynurenine (Km ¼ 3.0 lM± 0.10) and was inhibited by L-kynurenine (Ki ¼ 20 lM),
D-kynurenine (Ki ¼ 12 lM) and a synthetic substrate analogue D,L-3,7-dihydroxydesaminokynurenine (Ki ¼
100 nM) The activity/concentration profile for kynureninase from this source was sigmoidal in all instances There appeared to be partial inhibition by substrate, and excess pyridoxal 5¢-phosphate was found to be inhibitory Keywords: kynureninase; kynurenine; neuroprotection; quinolinic acid; tryptophan metabolism
Rapid progress in the pathophysiology of human diseases
has always been hampered by the availability of human
tissue, aesthetics, and ethical considerations The principle
aim of this study was to express a clone of human
kynureninase in an appropriate host that would yield
sufficient quantities of protein to permit identification and
biochemical characterization of the enzyme and
investiga-tion into the effects of various synthetic and endogenous
inhibitors Achieving these objectives could provide an
avenue for pharmacological modulation of the synthesis of
the N-methylD-aspartate receptor agonist and the
excito-toxin, quinolinic acid, in addition to elevating the levels of
the neuroprotective kynurenate [1] Quinolinic acid has been
implicated as an aetiological factor in a range of
neurode-generative diseases which include epilepsy, Huntington’s
disease, AIDS-related dementia, and septicaemia, where it is
released as part of the inflammatory response to injury [2,3]
Kynureninase is one of the enzymes involved in the
tryptophan metabolic pathway It is a pyridoxal
5¢-phos-phate (PLP)-dependent enzyme which catalyses the
b,c-hydrolytic cleavage of the amino acidsL-kynurenine (1) and
L-3-hydroxykynurenine (2) to giveL-alanine (3) and either
anthranilic acid (4) or 3-hydroxyanthranilic acid (5)
(Scheme 1) [4] This pathway is crucial in the biosynthesis
of nicotinamide nucleotides [5] and also gives rise to other
pathophysiologically important compounds such as picolinic acid, an enhancer of nitric oxide synthase expression [6] Kynureninase has been purified and characterized from a number of different sources, such as bacteria, vertebrates and fungi, but very little is known about the human form The microbial enzyme from some sources has been shown
to be present as two isozymes with differing specificities towardL-kynurenine and 3-hydroxykynurenine [7] Recom-binant human enzyme is reported to be a homodimer with a monomeric molecular mass of 52.4 kDa and shares an amino-acid sequence homology of about 85% with cyto-solic rat hepatic kynureninase, which has also been cloned and expressed [8] There have been a few attempts to isolate and purify cloned human kynureninase but with limited success, although the bacterial enzyme has been cloned [9] Previous researchers [10] have demonstrated activity in human embryonic kidney fibroblast (HEK-293)-transfected cell homogenates with a Kmof 13.2 lM for 3-hydroxyky-nurenine and 671 lMforL-kynurenine for the catalytically active human enzyme
M A T E R I A L S A N D M E T H O D S
Materials All chemicals (reagent grade) were purchased from Sigma except for the ion exchangers and the Affi-Blue gel, which were obtained from Bio-Rad The nitrocellulose filters used for concentration and buffer equilibration of active enzyme fractions were purchased from Millipore (UK)
Protein expression
A cDNA clone encoding human liver kynureninase was a gift from Dr Andrea Cesura, Hoffman la Roche The cDNA was isolated by the method of
Correspondence to N P Botting, School of Chemistry,
University of St Andrews, St Andrews, Fife KY16 9ST,
Scotland, UK Fax: + 44 1334 463808, Tel.: + 44 1334 463856,
E-mail: npb@st-andrews.ac.uk
Abbreviations: PLP, pyridoxal 5¢-phosphate.
Enzyme: kynureninase ( L -kynurenine hydrolase, EC 3.7.1.3).
7 (Received 27 November 2001, revised 25 February 2002, accepted
25 February 2002)
Trang 2[10] and cloned into the Bac-to-Bac Baculovirus
Expres-sion System (Gibco-BRL) which was used to express
kynureninase in Spodoptera frugiperda (Sf9) insect cells
[11] Sf9 cells were grown at 28C in TC-100 suspension
cultures of 10 L containing a minimal amount of fetal calf
serum (1–2%) until 10 mL of growing cells resulted in a
confluent layer when placed in a small Petri dish Infection
of the Sf9 cells and expression of kynureninase proceeded in
TC-100 in the absence of any fetal calf serum The infection
was allowed to proceed for 96 ± 12 h depending on the
degree of lysis Light microscopy was used to monitor the
infection process, and, when isolated nuclei appeared amid a
host of grossly deformed cells, the cells were harvested and
active enzyme extracted A purification protocol was then
developed (Table 1) to obtain homogeneous enzyme
Purification protocol
All steps were performed at 4C and long-term storage
occurred at )80 C The tubes for collecting the various
fractions always contained 20 lL of a 10-mM stock PLP
solution in order to increase the stability of kynureninase
The infected Sf9 cell culture (10 L) was harvested after 96 h
by centrifugation at 5000 g for 7 min Both the supernatant
and the pellet (whole cells) were retained Harvested insect
cells were resuspended in cold buffer A consisting of
100 mM Tris/HCl buffer (pH 7.5) containing 0.25M
sucrose, 1 mM dithiothreitol, 0.5 mMEGTA, 10 lMPLP,
100 lM phenylmethanesulfonyl fluoride, 2 lgÆmL)1
apro-tinin plus 1 lgÆmL)1pepstatin and leupeptin, and sonicated
on ice (All subsequent buffers contained the protease
inhibitors, PLP, dithiothreitol and EGTA at the above
concentrations.) The resultant fraction was then centrifuged
at 12 000 g for 20 min This procedure was repeated up to
four times with retention of the supernatant Both
supern-atants were shown to contain all the activity The
superna-tant was brought to 20% (NH4)2SO4saturation centrifuged
at 10 000 g for 15 min and the pellet discarded Then the
(NH4)2SO4 was increased to 80% to precipitate
kynuren-inase and centrifugation carried out as above This pellet
was redissolved in 3 mL buffer A, equilibrated with 50 mL
20 mM Tris/HCl at pH 8.6 and applied to a DEAE Blue Sepharose CL-6B affinity column (1.5· 30 cm) that had been equilibrated with 10 mMTris/HCl buffer at pH 8.6, at
a flow rate of 1.5 mLÆmin)1 The enzyme was eluted in the unbound fraction devoid of any PLP Concentration and equilibration of fractions containing active kynureninase during this step and elsewhere were performed in an Amicon ultrafiltration unit incorporating a nitrocellulose membrane of exclusion limit 50 kDa This system always retained the recombinant enzyme Fractions containing active enzyme were pooled, equilibrated in 10 mM potas-sium phosphate buffer (pH 7.2), concentrated to 3.0 mL, and applied to a hydroxyapatite (Ultrogel) column (3.0· 25 cm) equilibrated with 10 mM potassium phos-phate buffer at pH 7.2 The column with bound kynuren-inase was washed with 3 vol equilibration buffer and then eluted with a stepwise gradient of sodium phosphate (10–
500 mM) buffer at a flow rate of 1 mLÆmin)1 Kynureninase was eluted at 160 mM
Active fractions were again pooled, equilibrated in
20 mM Tricine/NaOH, pH 8.8, concentrated to 4.0 mL and applied to a strong anion-exchange (Macro-Prep strong
S support) column (1.5· 30 cm) previously equilibrated with 20 mMTricine/NaOH at pH 8.8 buffer at a flow rate
of 2 mLÆmin)1 Bound enzyme was washed with 3 column vol equilibration buffer followed by stepwise elution with NaCl (10–500 mM) in column equilibration buffer at a flow rate of 3.0 mLÆmin)1 The enzyme was eluted at 60 mM
(1.5· 30 cm) was equilibrated with 20 mM L-His/HCl buffer at pH 6.0, and the pooled active fractions from the anion-exchange step were equilibrated (20 mM L-His/HCl,
pH 6.0) and concentrated (4.0 mL) as previously The concentrated fraction was applied to the column at a flow rate of 2.0 mLÆmin)1, and bound kynureninase was washed with 3 column vol equilibration buffer and eluted stepwise with KCl (0–400 mM) in 20 mM L-His/HCl buffer, pH 6.0,
at a flow rate of 3.0 mLÆmin)1 Kynureninase was eluted at
60 mM KCl Samples containing kynureninase were pooled, concentrated (4.0 mL), and equilibrated in buffer (20 mMimidazole/HCl, pH 6.8) as described previously and applied to the strong anion-exchange column (1.5· 30 cm) used earlier at a flow rate of 2.0 mLÆmin)1 This column had been equilibrated with 20 mM imidazole/HCl buffer,
pH 6.8 The enzyme was eluted in the unbound fraction and was pooled, concentrated, and equilibrated in assay buffer (10 mMTris/HCl, pH 7.9), divided into aliquots, and stored at)80 C until further use The various purification steps were followed by SDS/PAGE (12% gels) [12]; where a Table 1 Fractional purification of recombinant human kynureninase from the supernatant of virus-infectedinsect (Sf9) cells Specific details are outlined in the text All activity assays were performed with 3-hydroxykynurenine as substrate and at saturating PLP.
Step
Total protein (mg)
Total activity (nmolÆmin)1)
Specific activity (nmolÆmin)1Æmg)1)
Fold purification
% Yield
Scheme 1.
Trang 3tryptic mass fingerprint obtained by matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) MS of
a band of the expected molecular mass confirmed its identity
as kynureninase The protein concentrations were
deter-mined with the Bradford assay [13] Recombinant human
kynureninase from the final anion-exchange step was
assayed for purity using both SDS/PAGE and
MALDI-TOF MS of the whole protein Both purified and crude
samples of recombinant kynureninase can be stored in the
absence or presence of PLP for extended periods of time
(> 12 months) at)80 C without any loss of activity At
4C the enzyme is stable for up to 2 weeks in the presence
of PLP, and this is how crude enzyme solution was stored
between successive steps
Activity assays
Kynureninase activity of the enzyme was monitored
spec-trofluorimetrically at 37C, with excitation of the product
3-hydroxyanthranilate at 330 nm and emission at 410 nm
and 310 nm and 417 nm respectively for anthranilate, by
the method of Shetty & Gaertner [7] A Perkin–Elmer
luminescence spectrometer (model LS50B) connected to a
Grant circulating water bath was used for this purpose The
final reaction volume was 3.0 mL consisting of 25 nmol
PLP (saturating), 10 mM potassium phosphate buffer at
pH 7.9, substrate 3-hydroxykynurenine, and an appropriate
volume of enzyme Enzyme was always added last for all
reactions including the inhibition studies The amount of
product formed was determined with reference to a
standard curve of fluorescence intensity against
3-hydroxy-anthranilate concentration The kinetic assays were
per-formed using both crude and pure (> 95%) forms of the
enzyme Reproducibility of the experimental findings was
confirmed with enzyme from different batches and varying
degrees of purity in addition to replicates from within the
same batch, and kinetic analyses showed no significant
difference between the various extracts A progress curve
was constructed to confirm the linear relationship between
product formation, protein concentration, and time
Lin-earity of the enzymatic reaction was determined over 5 min
To achieve temperature equilibration (37C), the assay
mixture was incubated for at least 5 min before initiation of
the reaction Graphs were plotted using theCRICKETGRAPH
and GraphPadPRISM3 software packages, and the kinetic
parameters Km and Vmax were obtained using non-linear
regression Lineweaver–Burk and Dixon [14] plots were
used to characterize the type of inhibition Hill analysis was
also carried out to confirm the co-operativity
R E S U L T S
Using the purification protocol described above, human
recombinant kynureninase was purified > 60-fold from the
supernatant fraction to yield active enzyme with a final
specific activity of 164 nmolÆmin)1Æ(mg protein))1 The full
results of the purification are given in Table 1
It is not known why there is an initial increase in the
activity during the purification (Table 1) given that the
purification was performed at saturating PLP
concentra-tions, however, there was no change in the electrophoretic
mobility of the SDS/mercaptoethanol-treated enzyme from
these crude extracts It is possible that an inhibitor molecule
has been removed during the purification procedure Disc-gel electrophoresis in the absence of reducing agents SDS and mercaptoethanol to determine the native dimeric molecular mass resulted in the appearance of two bands
at 52.5 and 95 kDa (gel image not shown), and this shows that the native protein exists mainly in the dimeric form There was, however, a fair amount of tailing between the two bands which was probably due to the continuous association and disassociation of the respective subunits Owing to the asynchronized viral infection cycle, lysis of a proportion of the transformed insect cells occurred, as was observed microscopically This resulted in the presence of exogenous active kynureninase in the tissue culture medium Hence an 80% (NH4)2SO4precipitation was performed on the supernatant obtained from harvesting the whole cells This fraction was purified separately, and the overall yield was significantly lower than the whole cell fraction but sufficient to warrant purification The total pooled (super-natant + whole cells) enzyme activity from 10 L culture medium was 14 lmolÆmin)1 with a specific activity of
164 nmolÆmin)1Æmg)1 (see Table 1 for fractional purifica-tion of the supernatant) The purified enzyme was shown to
be purified to homogeneity (Fig 1) by SDS/PAGE on a 12% gel with subsequent Coomassie Brilliant Blue staining The molecular mass as determined by MALDI-TOF MS was 52.4 kDa, which is > 99.99% that of the predicted amino-acid sequence encoded by the cloned 1600 bp A tryptic mass fingerprint obtained by MALDI-TOF MS also confirmed the identity of the protein as kynureninase The
UV absorption spectrum of the purified dimeric native protein at a concentration of 1.80 mgÆmL)1in 10 mMTris/ HCl buffer at pH 7.9 and 4C showed a peak at 432 nm, which was due to the presence of the PLP cofactor (data not shown) This scan was identical with that obtained by Kishore [15] for kynureninase from Pseudomonas marginalis
2
Kinetic characterization of the recombinant human kynureninase revealed that the enzyme was specific for 3-hydroxykynurenine with an experimentally observed Km
of 3.0 ± 0.10 lMfor the racemic substrate (D,L -3-hydroxy-kynurenine) [8,10] Graphical analysis shows that the
Fig 1 Analysis of purifiedrecombinant human kynureninase SDS/ PAGE (12% gel) of the purified supernatant fraction showing kynu-reninase (30 lg) at 52.4 kDa in the presence of PLP The gel was run as described by Laemmli [11] Standards were Sigma prestained SDS molecular mass markers (SDS-7B) in sample buffer containing 4% SDS and 10% 2-mercaptoethanol.
Trang 4enzyme is subjected to substrate regulation (graph not
shown) and responds to both substrate and inhibitors in a
sigmoidal fashion (Fig 2) In contrast to previous reports,
no substrate activity could be detected withL-kynurenine,
using either a fluorimetric or UV spectroscopic assay
However, L-kynurenine was found to be a competitive
inhibitor at low substrate concentrations (Ki ¼ 20 lM)
and non-competitive at higher levels of substrate
(Ki¢ ¼ 55 lM) (Fig 3).D-Kynurenine was also found to
inhibit the enzyme (data not shown, Ki ¼ 12 lM) as did a
novel synthetic analogue, D,L -3,7-dihydroxydesaminoky-nurenine [16] (Scheme 2), with a Kiof 100 nM The latter two compounds were also found to be mixed inhibitors of the enzyme (data not shown)
The pH optimum was determined as 8.25 and the activity
of the enzyme was found to be strongly dependent on the ionic strength of the buffer All assays, however, were performed at pH 7.9 because initial experiments with crude fractions were performed before the establishment of the pH-dependence curve There was no significant difference in terms of reaction velocity between these two pH values At
pH 7.9, in 10 mMTris/HCl buffer at 37C, velocity against substrate plots in the absence and presence (Fig 2) of the inhibitor D,L-3,7-dihydroxydesaminokynurenine (100 nM) were all distinctly sigmoidal, as was the percentage inhibition graph obtained withL-kynurenine (Fig 4) A reciprocal plot
of the data acquired for theD,L -3,7-dihydroxydesaminoky-nurenine-inhibited enzyme clearly reveals a highly co-operative enzyme throughout the whole substrate range, with negative co-operativity at low concentrations, which becomes positive as the substrate levels are increased (data not shown) Similar results were obtained by Hill analysis
D I S C U S S I O N
The results describe the first purification of human recom-binant kynureninase to homogeneity The protein was fully
Fig 2 Kynureninase activity as a function of 3-hydroxykynurenine ([s])
in the absence (,) andpresence [160 n M (h) and5 l M (n)
3,7-dihydroxydesaminokynurenine Run in 10 l M Tris/HCl buffer
(pH 7.9) Data are mean values of three replicate experiments, and the
assay was performed as described in the text.
Fig 4 Inhibition of kynureninase activity by L -kynurenine as a function
of 3-hydroxykynurenine concentration Data obtained at 10 l M Tris/ HCl at pH 7.9, 37 C and 15 l M substrate concentration (d) The depicted kynureninase inhibition is expressed as the percentage of the inhibition with reference to the activity observed in the absence of inhibitor.
Scheme 2.
Fig 3 Mixedinhibition of recombinant human kynureninase by
L -kynurenine Competitive inhibition (K i ¼ 20 l M ) observed at low
concentrations of substrate which becomes mixed (K i ¢ ¼ 55 l M ) at
higher levels of substrate in 10 m M Tris/HCl buffer at pH 7.9 and
37 C, K m ¼ 3.0 l M , specific activity of 164 nmolÆmin)1Æ(mg
pro-tein))1and n ¼ 3 Concentrations of L -kynurenine in l M were 0 (j),
16 (n), 32 (.), 64 (e), 128 (d) and 256 (h).
Trang 5characterized by electrophoresis (Fig 1)
and UV absorption spectroscopy, and the data are
consis-tent with previous reports [10,11] on the protein The kinetic
characterization revealed that the human recombinant
kynureninase is specific for 3-hydroxykynurenine, with a
Kmof 3.0 ± 0.1 lM This Km value is much lower than
previously reported in this [11] and other laboratories [6] for
the recombinant enzyme, and this is probably because our
findings are for an enzyme displaying sigmoidal kinetics and
thus the calculated Km is not the same as the Michaelis
constant Km but rather a sigmoidal constant Ks which
incorporates an interaction factor(s) and hence is not the
substrate concentration at 50% Vmax The data are,
however, consistent with values obtained for constitutive
enzymes isolated from other species such as Saccharomyces
cerevisiae(Km ¼ 3.0 lM) [17] and the fungus
roqueforti (Km ¼ 4.0 lM) [7] Soda & Tanizawa [18]
reported a Kmvalue of 1.67 lMfor rat hepatic
kynurenin-ase It was also not possible to show any activity towards
L-kynurenine, and, at the previously reported Kmvalues of
400 lM or more, there was significant inhibition of the
enzyme At a concentration of 250 lM L-kynurenine in the
presence of 625 nM D,L-3-hydroxykynurenine, there was
nearly 80% inhibition (Fig 4) This is a major difference of
human kynureninase from other mammalian enzymes, such
as rat hepatic kynureninase, and may imply that previous
reports of weak activity with L-kynurenine in crude cell
homogenates may be the result of additional adventitious
enzyme activity Certainly the preference for
3-hydroxyky-nurenine must be taken into account in inhibitor design Rat
hepatic kynureninase, on the other hand, showed activity
towardsL-kynurenine, with a Kmof 500 lMfor the partially
purified (> 80%) enzyme Differential substrate specificity
for kynureninase from brain and liver in mice has been
demonstrated by Chiarugi et al [19] in vivo, and this raises
the possibility of the existence of two isoforms of the enzyme
as discussed by Toma et al [8]
The pH optimum of 8.25 is consistent with previously
reported experimentally determined [6] values, and the
optimum activity of the enzyme using this potassium
phosphate buffer system showed a strong dependence on
ionic strength Molarity increases above 10–20 mMresult in
a significant fall in activity, which progressively worsens as
the ionic strength is increased
On the basis of the sigmoidal velocity plots obtained in
the absence and presence ofD,L
-3,7-dihydroxydesaminoky-nurenine (Fig 2), the enzyme appears to be subjected to
co-operative modulation by the substrate
3-hydroxykynur-enine The mixed inhibition depicted by the Lineweaver–
Burk plot in Fig 3 corroborates this with its reduced Vmax
and increased Km The shape of the plot is consistent with
binding of the inhibitor to both the free enzyme (E) and the
enzyme–substrate complex (ES) [15] and hence it can be
inferred that an additional ligand-binding site must be present on the human enzyme
When the lines intersect above the x-axis then Ki< Ki¢, and when the lines intersect below the x-axis then Ki> K0i (in both instances the lines have to intersect to the left of the y-axis) The data obtained for L-kynurenine gave
Ki ¼ 20 lMand Ki¢ ¼ 55 lM, while theD-isomer (data not shown) showed very similar behavior
C O N C L U S I O N S
The work described shows that appreciable quantities of active recombinant human kynureninase can be obtained using a baculovirus/insect cell system followed by a straightforward purification protocol This provides a relatively simple and economical method of producing active enzyme for use in mechanistic and structural studies Characterization of recombinant human kynureninase shows that it is similar to the rat liver enzyme [4] in terms of molecular mass, pH optimum, Km and sensitivity to analogue inhibitors, but it also has some important differences The human enzyme seems to be completely specific for 3-hydroxykynurenine with no significant activity with kynurenine, as reported previously This may be important in inhibitor design Also the enzyme appears to
be subjected to substrate modulation, exhibiting sigmoidal kinetics This behaviour could be important in regulation of enzyme activity in vivo and consequent channeling of substrate 3-hydroxykynurenine down the tryptophan– kynurenine metabolic pathway
The purification of recombinant human kynureninase
to homogeneity has allowed crystallization trials to commence in our laboratory for elucidation of the X-ray crystal structure of the protein The information obtained should provide invaluable knowledge on the active site and also pave the way for co-crystallization of enzyme–substrate and/or enzyme–inhibitor complexes These should allow further mechanistic investigation of the catalytic reaction and hence facilitate subsequent design and synthesis of effective inhibitors in an attempt
to combat the deleterious effects of the many serious neurodegenerative disorders
A C K N O W L E D G E M E N T S
A fellowship to H A W from the Wellcome Trust provided the funds for this study Dr C H Botting is acknowledged for helpful discussions, performing the MALDI-TOF MS, and valuable compu-ting assistance.
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