2B Keywords Ginkgo biloba; ginkgotoxin; pyridoxal kinase; c-aminobutyric acid; pyridoxal phosphate Correspondence C.. It is shown that ginkgotoxin serves as an alternate substrate for th
Trang 1ginkgotoxin from Ginkgo biloba
Uta Ka¨stner1, Christian Hallmen2, Michael Wiese2, Eckhard Leistner1and Christel Drewke1
1 Institut fu¨r Pharmazeutische Biologie, Universita¨t Bonn, Germany
2 Pharmazeutisches Institut, Pharmazeutische Chemie, Endenich, Bonn, Germany
4¢-O-methylpyridoxine (MPN, ginkgotoxin; Fig 1) is a
neurotoxic compound that causes severe neuronal
dis-orders in mammals after ingestion Symptoms of this
poisoning called ‘gin-nan sitotoxism’ are mainly
epilep-tic convulsions, paralysis of the legs and loss of
con-sciousness [1] There are even reports of death due to
overconsumption of Ginkgo seeds, which are the main
source of ginkgotoxin [1] In addition to the seeds,
which accumulate the toxin, ginkgotoxin has also been
found in the leaves of Ginkgo biloba as well as in
rem-edies produced from leaf extracts [2,3] These remrem-edies
are used in the therapy of insufficient central and
per-ipheral blood flow [4]
Ginkgotoxin is structurally related to vitamin B6
and likely interferes with its biosynthesis, metabolism
or function It is for this reason that ginkgotoxin is considered to be a B6 ‘antivitamin’, as is 4¢-deoxypyri-doxine (DPN) [5] (Fig 1), a synthetic analogue of the
B6 vitamers pyridoxal (PL), pyridoxamine (PM) and pyridoxine (PN) The physiologically active B6 vitam-ers pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) are most important, because they participate in many enzymatic reactions including amino acid metabolism [6] and reactions involved in the synthesis of neurotransmitters (e.g dopamine, sero-tonin, norephedrine and c-aminobutyric acid) [7] One key reaction is the formation of c-aminobutyric acid
by decarboxylation of glutamate, catalysed by the two isoforms of glutamate decarboxylase, GAD65 and GAD67, which require PLP as a cofactor (Fig 2B)
Keywords
Ginkgo biloba; ginkgotoxin; pyridoxal kinase;
c-aminobutyric acid; pyridoxal phosphate
Correspondence
C Drewke, Institut fu¨r Pharmazeutische
Biologie, Rheinische
Friedrich-Wilhelms-Universita¨t Bonn, Nussallee 6, 53115 Bonn,
Germany
Fax: +49 228 733250
Tel +49 228 732563
E-mail: cdrewke@uni-bonn.de
Website: http://www.uni-bonn.de/pharmbio/
(Received 5 September 2006, revised
11 December 2006, accepted 15 December
2006)
doi:10.1111/j.1742-4658.2007.05654.x
Ginkgotoxin (4¢-O-methylpyridoxine) occurring in the seeds and leaves of Ginkgo biloba, is an antivitamin structurally related to vitamin B6 Ingestion
of ginkgotoxin triggers epileptic convulsions and other neuronal symptoms Here we report on studies on the impact of B6antivitamins including ginkgo-toxin on recombinant homogeneous human pyridoxal kinase (EC 2.7.1.35)
It is shown that ginkgotoxin serves as an alternate substrate for this enzyme with a lower Km value than pyridoxal, pyridoxamine or pyridoxine Thus, the presence of ginkgotoxin leads to temporarily reduced pyridoxal phos-phate formation in vitro and possibly also in vivo Our observations are dis-cussed in light of Ginkgo medications used as nootropics
Abbreviations
DPN(P), 4¢-deoxypyridoxine(phosphate); GAD, glutamate decarboxylase; MPN(P), 4¢-O-methylpyridoxine(phosphate), ginkgotoxin(phosphate); PKH, human pyridoxal kinase; PL(P), pyridoxal(phosphate); PM(P), pyridoxamine(phosphate); PN(P), pyridoxine(phosphate).
Trang 2[8,9] This reaction is crucial for maintaining the
bal-ance between c-aminobutyric acid, the main inhibitory
neurotransmitter, and glutamate, the main excitatory
neurotransmitter in the brain Because of a
disregula-tion of neuronal excitability, a decrease in the
c-ami-nobutyric acid level often is accompanied by epileptic
seizures after induction by antivitamin B6 agents [10]
Likewise, an imbalance between the levels of glutamate and c-aminobutyric acid has also been observed in rat brain after intoxication with ginkgotoxin [11] Thus, there is an obvious connection between the main symptoms of gin-nan sitotoxism and the disregulation
of c-aminobutyric acid metabolism This disregulation has been explained by reduced GAD activity [10] In
N
O
H
R
CH2OH
N
O H R
CH2O P
N
O
CHO
N
O
CHO
N
CH2OH O
H CHO
R = CH2NH2 or CH2OH
ATP (1)
PL
PL
PLP
ATP (4)
Pi (3)
A Interconversion of B6-vitamers
I Reactions of the salvage pathway
(1), (2)
II Reactions before (3) and after (4)
passage through a cell membrane
(e.g blood brain barrier)
B Metabolism of neurotransmitter
glutamate PLP γ −aminobutyric acid (GABA)
CO2 (5)
Fig 2 Reactions potentially affected by B6antivitamins (ginkgotoxin, deoxypyridoxine) (1,4), Pyridoxal kinase; (2), pyridoxine ⁄ pyridoxamine phosphate oxidase; (3), pyridoxal phosphatase; (5), glutamate decarboxylase.
Fig 1 B6 antivitamins ginkgotoxin (phosphate) and 4¢-deoxypyridoxine (phosphate).
Trang 3cases of intoxication with ginkgotoxin, the decreased
availability of c-aminobutyric acid was assumed to be
caused by an inhibitory effect of the toxin on one or
both of the GAD isoenzymes due to the structural
fea-tures of ginkgotoxin [12]
To clarify these assumptions, the influence of
gin-kgotoxin on both GAD isoenzymes was examined at
the enzymatic level [8] Although a significant decrease
in GAD65 activity, to 35% of initial values, was
observed when GAD was incubated with
4¢-O-methyl-pyridoxine 5¢-phosphate (ginkgotoxin phosphate,
MPNP; Fig 1), the concentration of ginkgotoxin
phos-phate at which this inhibition took place (IC50¼
2.7 mm) probably is too high to be reached under
phy-siological conditions Thus, the GAD isoenzymes could
be ruled out as direct targets for ginkgotoxin or its
phosphate in vivo [8]
However, PLP-dependent GAD can be influenced
indirectly by ginkgotoxin via the reduced availability
of enzyme cofactor due to an inhibition of enzymes
involved in PLP formation Whereas plants and most
microorganisms are able to biosynthesize vitamin B6,
mammals depend on the uptake of B6 vitamers and
their conversion to PLP Phosphorylated dietary
pre-cursors of PLP have to be dephosphorylated prior to
resorption in the intestine [13,14] Bound to albumin
vitamin B6 derivatives are then distributed via the
bloodstream They are phosphorylated in the liver to
their respective 5¢-phosphate esters by pyridoxal kinase
and eventually oxidized by pyridoxine⁄ pyridoxamine
phosphate oxidase [7] Both enzymes are part of the
vitamin B6 ‘salvage pathway’ (Fig 2A) PLP is then
transported in the blood to different organs However,
before passage through the blood–brain barrier
circula-ting PLP has to be dephosphorylated again by
mem-brane-bound phosphatases (Fig 2A) [13,14] Inside the
brain a rephosphorylation to PLP takes place, again
catalysed by pyridoxal kinase (Fig 2A) [7,14] As a
consequence, there is a requirement for ubiquitous
expression of the kinase in mammalian tissues [14,15]
In the case of inhibition of the enzyme by ginkgotoxin
(phosphate) the availability of cofactor not only for
GAD, but also for all other PLP-dependent reactions
involved in neurotransmitter and amino acid
metabo-lism would be decreased resulting in a total
physiologi-cal imbalance of metabolism, very likely accompanied
by diverse pathological symptoms Thus, human
pyrid-oxal kinase (PKH) obviously plays a crucial role in the
regulation of PLP homoeostasis To elucidate the role
of this important enzyme and the mode of action of
ginkgotoxin in more detail, we studied recombinant
PKH as a possible target for ginkgotoxin
Results
Properties of PKH PKH catalyses the conversion of PL, PN and PM (Fig 2A) to the respective 5¢-phosphate esters using ATP as a cofactor [15,16] To examine the mode of action of ginkgotoxin on PKH, we overexpressed and purified the enzyme leading to a homogeneous protein
of the expected molecular mass as proven by SDS⁄ PAGE (data not shown) and by MALDI-TOF spectroscopy (see Supplementary material)
PKH was characterized with respect to its biochemi-cal properties The enzymatic activity was stable at )20 C for 21 days The enzyme was only active in the presence of ATP In contrast, no activity could be detected with GTP
Maximum activity was observed for a pH range 5.8–6.3 Accordingly, all measurements were performed
at pH 6.2 The velocity of the reaction showed a sharp optimum at 45C However, to create physiological conditions for determination of the effect of ginkgo-toxin on the human enzyme, further measurements were carried out at 37C
Identification of ginkgotoxin as a substrate
of PKH Incubation of PKH with ginkgotoxin in the presence of ATP gave a new compound, which during HPLC co-chromatographed with a synthetic sample of the 5¢-phosphate ester of ginkgotoxin and showed a time-dependent formation (see Supplementary Fig S2) Treatment of the product with alkaline phosphatase reversed the reaction: ginkgotoxin was formed at the expense of the newly detected compound indicating that the new metabolite was indeed identical to ginkgo-toxin phosphate (see Supplementary material) The experiment shows that the kinase phosphorylates not only B6 vitamers (Fig 2) but also ginkgotoxin Like-wise, incubations of PKH with DPN revealed that this synthetic antivitamin was also phosphorylated (data not shown)
In order to determine the kinetic data for PL, PN,
PM, MPN and DPN, two different test systems were employed: an HPLC assay and an optical test, in which phosphorylation of pyridoxal was measured (see Experi-mental procedures) The kinetic data given in Table 1 reveal that among all vitamin B6derivatives tested, the antivitamin DPN is the substrate with the highest maxi-mum velocity (2.62· 10)6nmolÆmg)1Æmin)1) and the highest turnover number (kcat¼ 1.535 s)1), whereas the
Trang 4antivitamin ginkgotoxin exhibits the highest affinity
(lowest Km¼ 4.95 · 10)6m) towards the PKH enzyme
(Table 1) This shows that the substituent at C-4¢ of the
pyridine ring system plays an important role in
deter-mining the kinetic features of PKH However, the data
for the catalytic efficiency of ginkgotoxin and DPN are
of the same order of magnitude and reflect their
antivita-min character, as they are much higher compared with
those of the physiological substrates PL and PM
(Table 1)
The Km value for PL (58.7 lm; Table 1) is more
than 10-fold higher than that of PL (3.3 lm) after
expression of PKH cDNA in human embryonic kidney
cells [15] This can be explained by different expression
systems employed Furthermore, our determination of
the Km value was performed by HPLC and with a
homogeneous enzyme preparation, whereas Hanna
et al [15] used a cell homogenate to determine the Km
in a fluorometer
When both PL (0.025 mm) and ginkgotoxin
(0.025 mm) were enzymatically phosphorylated by
PKH in separate experiments, the natural substrate
(PL) was converted to its 5¢-phosphate (kcat¼
0.995 s)1) faster than was ginkgotoxin (kcat¼
0.460 s)1) (Fig 3A) Coincubation of PL and
ginkgo-toxin, both at a concentration of 0.025 mm, however,
reversed the velocity of phosphorylation of both
sub-strates with PL being an almost inactive substrate within 14 min of start of the reaction (Fig 3B) Coin-cubation of both substrates with a reduced concentra-tion of ginkgotoxin (0.0125 mm), compared with PL (0.025 mm) still gave initially a faster phosphorylation
of ginkgotoxin than of PL (Fig 3C) Note that in this experiment a shorter incubation time (< 2 min) was not possible for technical reasons (Experimental proce-dures)
In these experiments the incubation mixtures were analysed by HPLC Supporting evidence for a relat-ively fast conversion of ginkgotoxin was obtained using the optical assay which detects PLP alone When
PL was kept constant (0.05 mm), and increasing con-centrations of ginkgotoxin (0–0.25 mm) were added, formation of PLP by PKH was more and more delayed depending on the concentration of ginkgotoxin (Fig 4A) However, after an initial ‘lag-phase’, PLP is formed with the same velocity as in the control with-out ginkgotoxin With high concentrations of ginkgo-toxin (0.25 mm) formation of PLP is suppressed completely during the incubation period (Fig 4A) For comparison, we performed the same assay with increasing concentrations of DPN [5] (Fig 1) The presence of DPN revealed a clear decrease in PLP for-mation Although the reaction velocity decreased no lag phase appeared in the presence of DPN (Fig 4C)
time [min]
0 2 4 6 8 10 0 2 4 6 8 10 12 14
formation of 5'-phosphate [nmol/ml] formation of 5'-phosphate [nmol/ml] formation of 5'-phosphate [nmol/ml]
2
4
6
8
formation of MPNP formation of PLP
time [min]
2 4 6 8
formation of MPNP formation of PLP
time [min]
2 4 6 8
formation of PLP formation of MPNP
Fig 3 Formation of pyridoxal 5¢-phosphate and ginkgotoxin phosphate (MPNP) during incubation of pyridoxal kinase separately (A) and simul-taneously (B,C) with pyridoxal and ginkgotoxin (A,B) PL ¼ 0.025 m M , ginkgotoxin ¼ 0.025 m M (C) PL ¼ 0.025 m M , ginkgotoxin ¼ 0.0125 m M Reactions were monitored using HPLC The data are the mean of two independent experiments.
Table 1 Kinetic data of different B 6 vitamers and antivitamins accepted as substrates by PKH.
Compound Km ( M ) V max (nmolÆmg)1Æmin)1) k cat (s)1) k cat ⁄ K m (mol)1Æs)1) K i ( M )
Trang 5Evidently, PLP formation by pyridoxal kinase follows
different kinetics in the presence of ginkgotoxin when
compared with DPN
The inhibitor constants (Table 1) for both
antivita-mins as determined using the optical method revealed
a significantly lower Kifor ginkgotoxin (4.14· 10)7m)
than DPN (5.74· 10)5m) It should be noted that the
Kivalue for ginkgotoxin was determined for the initial
linear range of the plot (Fig 4A)
Enzymatic assays with a constant concentration of
ginkgotoxin (0.2 mm) and variable concentrations of
PL (0.05–1.00 mm) demonstrated that the inhibitory
effect of ginkgotoxin on PKH can be alleviated With
increasing concentrations of PL, PLP was formed
with an increasing velocity (Fig 4B) The same is true
for the influence of DPN on the kinase When DPN
was kept constant (0.05 mm) and PL was added in
increasing concentrations (0.01–0.15 mm), an increase
in the velocity of PLP formation was observed
(Fig 4D)
From these results we conclude that both ginkgotoxin
and DPN compete with PL and that in the presence of
ginkgotoxin phosphorylation of PL is severely delayed due to the low Kmvalue of ginkgotoxin
Hydropathy of PKH and ginkgotoxin Because the affinity of ginkgotoxin for the PKH enzyme might be influenced by the lipophilicity of its substrates, the logP value, which is the decadic log-arithm of its distribution coefficient in an organic phase and the aqueous phase (PO⁄ W), was determined For ginkgotoxin a logP value of )0.299 was found This is significantly higher than the logP value deter-mined for PL ()1.182), demonstrating the higher lipo-philicity of the toxin
The preferred use of ginkgotoxin as a substrate by pyridoxal kinase was further analysed in detail by investigating the hydrophobicity distribution of the enzyme and substrates
Deduced from the crystal structure of pyridoxal kin-ase from sheep [17], the hydrophobic and hydrophilic regions of the substrate and cofactor binding domain
of PKH were modelled (see Experimental section)
0
2
4
6
8
10
12
14
16
20
22
24
26
time [min]
without MPN MPN 0.010 m M
MPN 0.015 m M
MPN 0.020 m M
MPN 0.025 m M
MPN 0.035 m M
MPN 0.040 m M
MPN 0.045 m M
MPN 0.050 m M
MPN 0.250 m M
A
0 10 20 30
time [min]
without MPN
PL 1.00 m M
PL 0.75 m M
PL 0.50 m M
PL 0.40 m M
PL 0.30 m M
PL 0.25 m M
PL 0.20 m M
PL 0.15 m M
PL 0.10 m M
PL 0.05 m M
B
time [min]
0
10
20
DPN 0.01 m M
DPN 0.05 m M
DPN 0.10 m M
DPN 0.20 m M
DPN 0.30 m M
C
0 20 40 60
time [min]
without DPN
PL 0.05 m M
PL 0.10 m M
PL 0.05 m M
PL 0.04 m M
PL 0.03 m M
PL 0.02 m M
PL 0.01 m M
D
Fig 4 Reversible inhibition of PKH by ginkgotoxin and 4¢-deoxypyridoxine during formation of pyridoxal phosphate (A) Inhibition by increas-ing amounts of MPN (ginkgotoxin) in the presence of PL PL ¼ 0.05 m M (B) Reversion of MPN (ginkgotoxin) caused inhibition by increasing amounts of PL MPN (ginkgotoxin) ¼ 0.2 m M (C) Inhibition by increasing amounts of DPN PL ¼ 0.05 m M (D) Reversion of DPN caused inhibition by increasing amounts of PL DPN ¼ 0.05 m M
Trang 6Figure 5 shows the enzyme’s substrate-binding domain
with ginkgotoxin and the terminal phosphate group of
ATP The most lipophilic part of the active site of the
enzyme is located close to the methyl ether group of
ginkgotoxin (brown colour) It is built up of
hydropho-bic side chains of two tyrosine residues This additional
hydrophobic interaction is in agreement with the higher
affinity of ginkgotoxin to PKH in comparison with PL
Discussion
The active forms of vitamin B6 are PLP and PMP,
cofactors involved in amino acid and neurotransmitter
metabolism [6,7] For their formation, phosphorylation
of unphosphorylated dietary precursors catalysed by
PKH is required ubiquitously in mammalian tissues
[15] Decreased activity of PKH leads inter alia to a
decreased availability of PLP for GAD, which
cata-lyses the formation of c-aminobutyric acid, the most
potent inhibitory neurotransmitter in the mammalian
brain Decreased GAD activity in turn leads to an
imbalance between excitatory and inhibitory
neuro-transmission, which may result in epileptic convulsions
[18] In this context, PKH appears to be a plausible
target for the antivitamin ginkgotoxin, which was
shown to trigger epileptic seizures in mammalia [1]
To date, the effect of ginkgotoxin on pyridoxal
kin-ase had been studied only with a partially purified
homogenate from mouse brain [5] Detailed
experi-ments on the mode of inhibition of the human enzyme
by ginkgotoxin are lacking This study shows for the first time an enzymatic conversion of ginkgotoxin to ginkgotoxin phosphate and of DPN to DPNP by homogeneous human pyridoxal kinase Because all three physiological B6 vitamers are also converted by the purified enzyme in our assay, the term ‘pyridoxal kinase’ may be changed to ‘PN⁄ PL ⁄ PM kinase’ in agreement with the term suggested for the PN, PL and
PM converting enzyme in Escherichia coli [19]
The conversion of ginkgotoxin and DPN to ginkgo-toxin phosphate and DPNP, respectively, demonstrates that both antivitamins, like the vitamers, are substrates
of the enzyme Thus, the kinase acts on two competing substrates, when PL and one of the two antivitamins are simultaneously employed in the enzymatic assay This is evident from Fig 4B,D, which show that the inhibitory effect of ginkgotoxin and DPN on PLP for-mation can be alleviated by increasing amounts of PL
In the case of PL coincubated with ginkgotoxin, a lag phase of PLP formation is observed The duration of this lag phase depends on the concentration of ginkgo-toxin (Fig 4A) and is alleviated by the addition of PL (Fig 4B) This interesting mixed-substrate phenom-enon is characteristic of a substrate in the presence of another substrate with a higher affinity and at the same time a lower maximum velocity and turnover than the substrate being tested [20,21]
Figure 4A,C shows that PLP formation by PKH fol-lows different kinetics in the presence of either ginkgo-toxin or DPN Furthermore, the Ki values determined for both antivitamins are significantly different (Table 1), showing that ginkgotoxin is a significantly stronger inhibitor than DPN Hanna et al reported that the type of PKH inhibition by DPN after expres-sion of the respective gene in human embryonic kidney cells is competitive [15] This is in agreement with our observations
Comparing the values for the catalytic efficiency (Table 1), it is evident that ginkgotoxin (9.30· 104 mol)1Æs)1) and DPN (7.10· 104 mol)1Æs)1) are phos-phorylated more efficiently than PL (1.70· 104 mol)1Æs)1) and PM (6.28 · 103mol)1Æs)1) by PKH This reflects the antivitamin character of both compounds and explains a depletion of cofactor formation in the organism in the presence of the antivitamins
The kinetic data (Table 1) are a reflection of the structural features of ginkgotoxin, PL and PKH LogP values determined for PL ()1.182) and ginkgotoxin ()0.299) show a higher lipophilicity for ginkgotoxin in comparison with the natural substrate This is in agree-ment with the molecular structure of ginkgotoxin, which because of its 4¢-O-methyl group is more hydro-phobic in comparison with the B6 vitamers (Fig 1)
Fig 5 View into the active site of PKH The hydrophobic
proper-ties are colour coded from hydrophobic (brown) to more hydrophilic
(green) Shown are one phosphate residue of ATP and ginkgotoxin
as docked using the program GOLD
Trang 7The lipophilicity of ginkgotoxin is in line with the
properties of the enzyme’s substrate-binding domain,
which owing to its hydrophobicity (Fig 5) prefers a
lipophilic over a hydrophilic substrate
The lipophilic toxin very likely passes the blood–
brain barrier more easily than the B6 vitamers This
may contribute to an increased interaction of
ginkgo-toxin with PKH in vivo
It should be emphasized that the toxin has been
reported to also be present in different Ginkgo
remed-ies [2,3], which are top-selling phytotherapeutic
medi-cations in Europe [22] Strikingly, two cases of
recurrence of well-controlled epilepsy after ingestion of
Ginkgo biloba remedies by two elderly patients have
been reported recently [23] After the immediate
with-drawal of the Ginkgo remedy, in both cases no further
epileptic convulsions were stated within the
observa-tion interval (8 months, first patient, 4 months, second
patient) According to another publication, several
other cases of seizure associated with Ginkgo have
been reported [24] The authors of these case studies
assume that the occurrence of the epileptic convulsions
was due to the ingestion of Ginkgo remedies However,
they neither specify the respective remedies, nor do
they mention their composition Therefore, the
possi-bilities of overdosage or interactions with other
medi-cations cannot be excluded However, Ginkgo biloba
extracts have been reported to have a proconvulsive
activity on chinchilla rabbits [25], and it should also be
mentioned that the action of GABAergic antiepileptica
was negatively influenced, when seizures were induced
in mice by various toxins [26] Thus, the question
ari-ses whether the occurrence of seizures really could be
due to the presence of ginkgotoxin when Ginkgo
rem-edies are ingested, at least by predisposed patients An
explanation may be derived from a comparison of the
vitamin B6 concentration in human blood and the
gin-kgotoxin concentration in human blood after ingestion
of Ginkgo remedies The maximum daily intake of
gin-kgotoxin in remedies based on Ginkgo extract was
cal-culated to 58.62 lg [2] Accordingly, the maximum
concentration of ginkgotoxin in human plasma
calcula-ted for 6 and 4 L of blood should be in a range of
53.33–80.24 nm, provided that the toxin is
distri-buted exclusively in the blood This is in the same
order of magnitude for vitamin B6 levels in plasma,
which is reported to be 114 nm [27] Thus, due to its
high affinity to PKH, ginkgotoxin may directly
inter-fere with the enzyme not only in vitro but also in vivo
Unpublished data from this laboratory show that
pyridoxine phosphate oxidase [28] and pyridoxal
phos-phatase [14], two other enzymes involved in vitamin B6
metabolism, are not inhibited by ginkgotoxin or its
phosphate This has also been experienced for GAD65 and GAD67, at least when physiologically relevant concentrations of the toxin were tested in vitro [8] It follows that the interaction between ginkgotoxin and PKH is a rather specific process which affects the key reaction for the supply of the human brain with PLP
Experimental procedures
Cloning and expression of PKH The sequence of PKH [15] was amplified by PCR according
to a standard protocol using vector pCDM8-PKH [15] as template The amplification product was proven to be free of any mismatches and inserted into vector pET11a (Novagen,
(rB– mB–) gal dcm (DE3)] was then transformed with the resulting recombinant vector pET11a-PKH The recombin-ant strain BL21 (D3) (pET11a-PKH) was grown in Luria–
37C until D600¼ 0.5 Isopropyl thio-b-d-galactoside was added to a final concentration of 1.0 mm, and the culture was incubated with shaking for 24 h Protein expression was
1 L of culture was resuspended in 10 mL of column buffer
frozen bacterial cells were thawed in a cold water bath before ultrasonic treatment (Branson Sonifier, Danbury, MA, 10·,
10 s, 50% output at stage 5) After sedimentation of the cell debris (30 min, 9000 g, 4C) the supernatant was treated as described below
Purification of PKH Cell-free protein extract derived from 6 L of culture was adjusted to 100 mm KCl and further successively subjected
to affinity chromatography (matrix:
Ger-many, prepared as recommended by the manufacturer) and gel filtration as described by Cash et al [30] The protein concentration in the eluted fractions was measured and the
[29] and MALDI-TOF-MS (see Supplementary material)
General analytical methods
MPN(P) was detected using HPLC as described previously
for Windows Integration Package (Knauer GmbH, Berlin, Germany)
To prove the enzyme’s identity and purity, pyridoxal kin-ase was subjected to MALDI-TOF-MS The analysis was
Trang 8conducted on a TofSpec E apparatus (Micromass,
Manches-ter, UK) in the group of K Sandhoff (Kekule-Institut fu¨r
Organische Chemie und Biochemie, Universita¨t Bonn,
Ger-many) The matrix was prepared as follows: 10 mg of
acetonitrile containing 0.1% trifluoroacetic acid The method
was performed under reflection in the positive mode
Determination of the logP values for ginkgotoxin
and PL
The determination of the logP values was performed
according to OECD guidelines for the testing of chemicals,
partition coefficient (n-octanol⁄ water): Shake Flask Method
(adopted by the council on 27 July 1995)
Enzyme incubation
All enzyme incubations carried out to determine activity of
pyridoxal kinase were performed in 70 mm potassium
phos-phate buffer The pH was adjusted to the optimum
(pH 6.2) as determined for the enzyme The total volume of
the incubation samples was 1 mL containing 10 lL each of
determine the temperature and pH optima, reactions were
8.0, respectively
incu-bated for 3 min with PL (0.005–0.5 mm), for 1 min with PN
(0.005–0.1 mm), for 1 min with PM (0.05–0.4 mm), for
1 min with DPN (0.015–0.1 mm) and for 30 s with
ginkgo-toxin (0.005–0.025 mm), respectively Incubation was
ter-minated by immediate injection of a sample (50 lL) of the
were determined according to Lineweaver–Burk plots and
calculated using software grafit v 5.0 (Erithacus Software
Ltd, Horley, UK) With pyridoxal as substate activity of
pyridoxal kinase was determined in parallel by monitoring
the increase in absorbance at 388 nm (absorption maximum
Mu¨nchen, Germany) The incubation period varied between
10 and 40 min depending on the concentration of substrate
The initial velocity data were fitted using grafit v 5.0
soft-ware This method provided a useful confirmation of the
val-ues obtained by HPLC All assays were generally performed
in triplicate
Molecular docking of ginkgotoxin at the active
site of PKH
The 3D structure of PKH was obtained from the Protein
Data Bank PDB-ID: 2AJP (S Ismail, S Dimov, A
Atan-assova, W.M Tempel, C Arrowsmith, A Edwards,
M Sundstrom, J Weigelt, A Bochkarev & H Park, unpublished manuscript) It is based on X-ray diffraction with a resolution of 2.5 A˚ The 3D structure of ginkgotoxin was generated using the sybyl sketch module (Tripos Inc.,
St Louis, MO), with subsequent force field minimization (MMFF94s force field; MMFF94 charges; termination cri-terion: gradient 0.005 kcalÆ(mol*A))1 Docking experiments were performed using gold 3.0.1 (Cambridge Crystallo-graphic Data Centre, Cambridge, UK) The active site was defined as all protein atoms within 10 A˚ distance from the sidechain oxygen of Ser12 The number of GA runs was set
to 30, otherwise the standard default settings were used In the initial docking results the 4¢-O-methyl group of ginkgo-toxin was located near Gly20 Comparing this result with the crystal structure of PL cocrystalized with sheep pyrid-oxal kinase [17] (PDB-ID: 1RFU), ginkgotoxin was turned through 180 These first results make no sense, because in this way it is not possible to phosphorylate the hydroxyl group at position 5 of MPN as experimentally observed (see below) Therefore we set distance constraints for the docking algorithm The distance between the carbon of the
to be between 5 and 7 A˚ (with a spring constant of 5.0) The distance between these two atoms is 6.0 A˚ in the crys-tal structure of sheep pyridoxal kinase
Acknowledgements
This study was supported by the Deutsche Fors-chungsgemeinschaft (Graduiertenkolleg GRK 677:
‘Struktur und molekulare Interaktion als Basis der Arzneimittelwirkung’) We are grateful to Dr Ewen Kirkness, Institute for Genomic Research, Rockville,
MS for providing us with a clone of human pyridoxal kinase and to Dr Thomas Hemscheidt, University of Hawaii at Manao, Honolulu, HI for providing us with
a synthetic sample of 4¢-O-methylpyridoxine
References
1 Wada K, Ishigaki S, Ueda K, Sakata M & Haga M (1985) An antivitamin B6, 4¢-methoxypyridoxine, from the seed of Ginkgo biloba L Chem Pharm Bull 33, 3555–3557
2 Arenz A, Klein M, Fiehe K, Groß J, Drewke C, Hemsc-heidt T & Leistner E (1996) Occurrence of neurotoxic 4¢-O- methylpyridoxine in Ginkgo biloba leaves, Ginkgo medications and Japanese Ginkgo food Planta Med 62, 548–551
3 Scott PM, Lau BP, Lawrence GA & Lewis DA (2000) Analysis of Ginkgo biloba for the presence of ginkgo-toxin and ginkgoginkgo-toxin 5¢-glucoside J AOAC Int 8, 1313–1320
Trang 94 Caesar W (1994) In ‘Ginkgo’, Urbaum und
& Schmoll H, eds), pp 23–31 Wissenschaftliche
Ver-lagsgesellschaft, Stuttgart, Germany
5 Mizuno N, Kawakami K & Morita E (1980)
Competi-tive inhibition between 4¢-substituted pyridoxine
ana-logues and pyridoxal for pyridoxal kinase from mouse
brain J Nutr Sci Vitaminol 26, 535–543
6 Coburn SP, Slominski A, Mahuren JD, Wortsman J,
Hessle L & Millan JL (2003) Cutaneous metabolism of
vitamin B-6 J Invest Dermatol 120 (2), 292–300
7 Ngo EO, LePage GR, Thanassi JW, Meisler N &
Nut-ter LM (1998) Absence of pyridoxine-5¢-phosphate
oxid-ase (PNPO) activity in neoplastic cells Isolation,
characterization, and expression of PNPO cDNA
Bio-chemistry 37, 7741–7748
8 Buss K, Drewke C, Lohmann S, Piwonska A & Leistner
E (2001) Properties and interaction of heterologously
expressed glutamate decarboxylase isoenzymes GAD
(65kDa) and GAD (67kDa) from human brain with
gink-gotoxin and its 5¢-phosphate J Med Chem 44, 3166–3174
9 Bu DF, Erlander MG, Hitz BC, Tillakaratne NJK,
Kaufman DL, WagnerMcPherson CB, Evans GA &
Tobin AJ (1992) Two human glutamate decarboxylases,
65- kDa and 67- kDa GAD, are each encoded by a
single gene Proc Natl Acad Sci USA 89, 2115–2119
10 Nitsch C & Okada Y (1976) Differential decrease of
GABA in the substantia nigra and other discrete regions
of the rabbit brain during the preictal period of
methox-ypyridoxine-induced seizures Brain Res 105, 173–178
11 Yanai A, Minami M, Takano Y, Endo T, Hamaue M,
Wada K, Take Y, Haga M, Morii K, Togashi H,
Yos-hioka M & Saito H (1990) 4¢-O-Methylpyridoxine
induced convulsion in guinea pigs and rats Life Sci Adv
107, 1499–1500
12 Wada K (2005) Ginkgo seeds food poisoning Chudoku
Kenkyu 18, 11–16
13 Merrill CR & Henderson JM (1990) Vitamin B6
metabo-lism by human liver Ann NY Acad Sci 585 (1), 110–117
14 Jang YM, Kim DW, Kang TC, Won MH, Baek NI,
Moon BJ, Choi SY & Kwon OS (2003) Human pyridoxal
phosphatase: molecular cloning, functional expression,
and tissue distribution J Biol Chem 278, 50040–50046
15 Hanna MC, Turner AJ & Kirkness EF (1997) Human
pyridoxal kinase, cDNA cloning, expression, and
modu-lation by ligands of the benzodiazepine receptor J Biol
Chem 272, 10756–10760
16 Di Salvo ML, Hunt S & Schirsch V (2004) Expression,
purification, and kinetic constants for human and
Escherichia coli pyridoxine kinases Protein Expr Purif
36 (2), 300–306
17 Li MH, KwokF, Chang WR, Lau CK, Zhang JP, Lo
SC, Jiang T & Liang DC (2002) Crystal structure of
brain pyridoxal kinase, a novel member of the
riboki-nase superfamily J Biol Chem 277 (48), 46385–46390
18 Tunnicliff G & Ngo TT (1998) Functional modification
of proteins of the nervous system by pyridoxal 5¢-phos-phate Cellular physiology and biochemistry Int J Exp Cell Physiol Biochem Pharmacol 8 (3), 117–129
19 Yang Y, Zhao G & Winkler ME (1996) Identification
of the pdxK gene that encodes pyridoxine (vitamin B6) kinase in Escherichia coli K-12 FEMS Microbiol Lett
141, 89–95
20 Willsta¨tter R, Kuhn R, Lind O & Memmen F (1927) U¨ber Hemmung der Leberesterase durch Ketocarbon-sa¨ureester Hoppe-Seyler’s Z Physiol Chem 167, 303–309
21 Dixon M & Webb EC (1979) Enzymes, 3rd edn 1979, Academic Press, New York, NY
22 Sticher O (1992) Ginkgo biloba – Analytik und Zuberei-tungsformen Pharm Unserer Zeit 6, 253–265
23 Granger AS (2001) Ginkgo biloba precipitating epileptic seizures Age Ageing 3, 523–525
24 Gregory PJ (2001) Seizure associated with Ginkgo biloba? Ann Intern Med 134 (4), 344
25 Pilija V, Ivetic V, Mihalj M, Draganic-Gajic S & Popo-vic M (2004) Effects of Ginkgo biloba extract on an experimental model of epilepsy Med Pregl 57 (11–12), 541–544
26 Anshu M, Pillai K & Zakir H (1996) Influence of Ginkgo biloba on the effect of anticonvulsants Indian
J Pharmacol 28, 84–87
27 Friedrich W (1988) Vitamins, pp 543–618 Walter de Gruyter, Berlin, New York, NY
28 Kang JH, Hong ML, Kim DW, Park J, Kang TC, Won
MH, Baek NI, Moon BJ, Choi SY & Kwon OS (2004) Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5¢-phosphate oxidase Eur J Biochem 271, 2452–2461
29 Laemmli UK (1970) Cleavage of structural proteins dur-ing the assembly of the head of bacteriophage T4 Nature 227, 680–685
30 Cash CD, Maitre M, Rumigny JF & Mandel P (1980) Rapid purification by affinity chromatography of rat brain pyridoxal kinase and pyridoxamine-5-phosphate oxidase Biochem Biophys Res Commun 96, 1755–1760
Supplementary material
The following supplementary material is available online:
Fig S1 MALDI-TOF mass spectrum of the recombi-nant homogeneous pyridoxal kinase The spectrum shows the singly charged (35035 Da) and the doubly charged (17470 Da) monomer as well as a singly charged dimer (70092 Da) and a minor amount of a doubly charged trimer (52694 Da)
Fig S2 (A) Formation of ginkgotoxin 5¢-phosphate (retention time: 7.5 min) from ginkgotoxin (retention time: 15 min) after incubation for 5 min (a) and
Trang 1060 min (b) with pyridoxal kinase (5 lg) at 37C, as
analyzed by HPLC (B) Formation of ginkgotoxin
(retention time: 15 min) from ginkgotoxin 5¢-phosphate
(retention time: 7.5 min) after treatment for 5 min (a)
and 60 min (b) with alkaline phosphase (Merck,
Darmstadt, Germany; 100 U) at 37C, as analyzed by
HPLC
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