The human pyridoxal kinase, a plausible target for ginkgotoxin from Ginkgo biloba Uta Ka ¨ stner 1 , Christian Hallmen 2 , Michael Wiese 2 , Eckhard Leistner 1 and Christel Drewke 1 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 remedies are used in the therapy of insufficient central and per- ipheral blood flow [4]. Ginkgotoxin is structurally related to vitamin B 6 and likely interferes with its biosynthesis, metabolism or function. It is for this reason that ginkgotoxin is considered to be a B 6 ‘antivitamin’, as is 4¢-deoxypyri- doxine (DPN) [5] (Fig. 1), a synthetic analogue of the B 6 vitamers pyridoxal (PL), pyridoxamine (PM) and pyridoxine (PN). The physiologically active B 6 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, GAD 65 and GAD 67 , 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 B 6 . Ingestion of ginkgotoxin triggers epileptic convulsions and other neuronal symptoms. Here we report on studies on the impact of B 6 antivitamins 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 K m 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). 1036 FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS [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 B 6 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 OH R CH 2 OH N OH R CH 2 O P N OH CH 2 O P CHO N OH CH 2 OH CHO N CH 2 OHOH CHO PM or PN PMP or PNP R = CH 2 NH 2 or CH 2 OH ATP FMN (1) (2) ATP (1) PL PL PLP ATP (4) Pi (3) I II I A Interconversion of B 6 -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 γ −aminobutyric acid (GABA) PLP CO 2 (5) Fig. 2. Reactions potentially affected by B 6 antivitamins (ginkgotoxin, deoxypyridoxine). (1,4), Pyridoxal kinase; (2), pyridoxine ⁄ pyridoxamine phosphate oxidase; (3), pyridoxal phosphatase; (5), glutamate decarboxylase. Fig. 1. B 6 antivitamins ginkgotoxin (phosphate) and 4¢-deoxypyridoxine (phosphate). U. Ka ¨ stner et al. Influence of ginkgotoxin on human pyridoxal kinase FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS 1037 cases 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 GAD 65 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 (IC 50 ¼ 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 B 6 , mammals depend on the uptake of B 6 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 B 6 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 B 6 ‘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 45 °C. However, to create physiological conditions for determination of the effect of ginkgo- toxin on the human enzyme, further measurements were carried out at 37 °C. 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 B 6 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 B 6 derivatives tested, the antivitamin DPN is the substrate with the highest maxi- mum velocity (2.62 · 10 )6 nmolÆmg )1 Æmin )1 ) and the highest turnover number (k cat ¼ 1.535 s )1 ), whereas the Influence of ginkgotoxin on human pyridoxal kinase U. Ka ¨ stner et al. 1038 FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS antivitamin ginkgotoxin exhibits the highest affinity (lowest K m ¼ 4.95 · 10 )6 m) 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 K m 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 K m value was performed by HPLC and with a homogeneous enzyme preparation, whereas Hanna et al. [15] used a cell homogenate to determine the K m 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 (k cat ¼ 0.995 s )1 ) faster than was ginkgotoxin (k cat ¼ 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 101214 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] 02040 2 4 6 8 formation of PLPformation of MPNP ABC 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 mM. (C) PL ¼ 0.025 mM, 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 )1 Æmin )1 ) k cat (s )1 ) k cat ⁄ K m (mol )1 Æs )1 ) K i (M) Pyridoxal 5.87 ± 0.28 · 10 )5 1.70 ± 0.36 · 10 )6 0.995 1.70 · 10 4 – Pyridoxine 9.87 ± 1.20 · 10 )6 2.11 ± 0.07 · 10 )6 1.233 1.25 · 10 5 – Pyridoxamine 1.26 ± 0.25 · 10 )4 1.35 ± 0.10 · 10 )6 0.790 6.28 · 10 3 – 4’-Deoxypyridoxine 2.16 ± 1.05 · 10 )5 2.62 ± 0.40 · 10 )6 1.535 7.10 · 10 4 5.74 · 10 )5 Ginkgotoxin 4.95 ± 0.21 · 10 )6 7.87 ± 0.69 · 10 )7 0.460 9.30 · 10 4 4.14 · 10 )7 U. Ka ¨ stner et al. Influence of ginkgotoxin on human pyridoxal kinase FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS 1039 Evidently, 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 K i for ginkgotoxin (4.14 · 10 )7 m) than DPN (5.74 · 10 )5 m). It should be noted that the K i value 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 K m value 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 (P O ⁄ 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 20 40 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 time [min] formation of pyridoxal-5'-phosphate [nmol/ml] without MPN MPN 0.010 m M MPN 0.015 mM MPN 0.020 mM MPN 0.025 mM MPN 0.035 mM MPN 0.040 mM MPN 0.045 mM MPN 0.050 mM MPN 0.250 mM A 01020 0 10 20 30 time [min] formation of pyridoxal-5'-phosphate [nmol/ml] without MPN PL 1.00 m M PL 0.75 mM PL 0.50 mM PL 0.40 mM PL 0.30 mM PL 0.25 mM PL 0.20 mM PL 0.15 mM PL 0.10 mM PL 0.05 mM B time [min] 0246810 formation of pyridoxal-5'-phosphate [nmol/ml] 0 10 20 30 without DPN DPN 0.01 mM DPN 0.05 mM DPN 0.10 mM DPN 0.20 mM DPN 0.30 mM C 0 20 40 60 time [min] 0246810 formation of pyridoxal-5'-phosphate [nmol/ml] without DPN PL 0.05 m M PL 0.10 mM PL 0.05 mM PL 0.04 mM PL 0.03 mM PL 0.02 mM PL 0.01 mM 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 mM. (D) Reversion of DPN caused inhibition by increasing amounts of PL. DPN ¼ 0.05 m M. Influence of ginkgotoxin on human pyridoxal kinase U. Ka ¨ stner et al. 1040 FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS Figure 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 B 6 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 B 6 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 K i 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 · 10 4 mol )1 Æs )1 ) and DPN (7.10 · 10 4 mol )1 Æs )1 ) are phos- phorylated more efficiently than PL (1.70 · 10 4 mol )1 Æs )1 ) and PM (6.28 · 10 3 mol )1 Æs )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 B 6 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. U. Ka ¨ stner et al. Influence of ginkgotoxin on human pyridoxal kinase FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS 1041 The 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 B 6 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 B 6 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 B 6 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 B 6 metabolism, are not inhibited by ginkgotoxin or its phosphate. This has also been experienced for GAD 65 and GAD 67 , 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, Madison, WI). E. coli strain BL21 (DE3) [F – ompT hsdS B (r B – m B – ) 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– Bertani medium containing penicillin G (100 lgÆmL )1 )at 37 °C until D 600 ¼ 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 confirmed by SDS ⁄ PAGE [29]. The cell pellet derived from 1 L of culture was resuspended in 10 mL of column buffer (20 mL of 1 m Tris ⁄ HCl, pH 7.4; 11.7 g NaCl, 2 mL of 0.5 m EDTA ad 1 L) and frozen overnight at )20 °C. The 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,4°C) 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: pyridoxyl–EAH–Seph- arose Ò 4B; Amersham Biosciences Europe, Freiburg, 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 purity of pyridoxal kinase was determined by SDS ⁄ PAGE [29] and MALDI-TOF-MS (see Supplementary material). General analytical methods Enzymatic formation of vitamin B 6 derivatives and MPN(P) was detected using HPLC as described previously [2]. Peaks were evaluated using the eurochrom TM 2000 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 Influence of ginkgotoxin on human pyridoxal kinase U. Ka ¨ stner et al. 1042 FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS conducted 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 sinapi- nic acid are vortexed in 1 mL of 60 : 40 (v ⁄ v) water ⁄ 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 a10mm ZnCl 2 stock solution and a 0.1 m ATP stock solu- tion. The general incubation temperature was 37 °C. To determine the temperature and pH optima, reactions were performed in a range from 20 to 60 °C and from pH 5.0 to 8.0, respectively. To determine K m values, 5 lg of purified PKH were 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 incubation mixture into the HPLC apparatus. K m values were determined according to Lineweaver–Burk plots and K i values according to Dixon plots. Regression lines were 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 of PLP) in a Uvikon Ò 810 spectrophotometer (Kontron, 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 2-methyl group and the C b -atom of Val41 was constrained 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 ginkgotoxin 5¢-glucoside. J AOAC Int 8, 1313–1320. U. Ka ¨ stner et al. Influence of ginkgotoxin on human pyridoxal kinase FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS 1043 4 Caesar W (1994) In ‘Ginkgo’, Urbaum und Arzneipflanze-Mythos, Dichtung und Kunst (Schmid M, & 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 Influence of ginkgotoxin on human pyridoxal kinase U. Ka ¨ stner et al. 1044 FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS 60 min (b) with pyridoxal kinase (5 lg) at 37°C, 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 37°C, as analyzed by HPLC. This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary material supplied by the authors. Any queries (other than missing material) should be directed to the corres- ponding author for the article. U. Ka ¨ stner et al. Influence of ginkgotoxin on human pyridoxal kinase FEBS Journal 274 (2007) 1036–1045 ª 2007 The Authors Journal compilation ª 2007 FEBS 1045 . The human pyridoxal kinase, a plausible target for ginkgotoxin from Ginkgo biloba Uta Ka ¨ stner 1 , Christian Hallmen 2 , Michael Wiese 2 , Eckhard. 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,