Original Paper 563

Influence of Antivitamins Ginkgotoxin 5′-Phosphate and Deoxypyridoxine 5′-Phosphate on Human 5′-Phosphate Oxidase*

Authors Nora Salamon, Cristian Gurgui, Eckhard Leistner, Christel Drewke

Affiliation Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany

Key words Abstract inhibited in vitro by the synthetic de- l" Ginkgo biloba ! rivative 4′-deoxypyridoxine 5′-phosphate but not l" Ginkgoaceae The pharmacological effects of leaf extracts (EGb by ginkgotoxin or its 5′-phosphate. l" ′‑ ginkgotoxin 5 phosphate 761) from Ginkgo biloba L. are attributed to gink- l" 4′‑deoxypyridoxine 5′‑phos- golides, bilobalide and biflavonoids. However, be- phate l" pyridoxine 5′‑phosphate oxi- sides these beneficial attributes, ginkgotoxin, a B6 Abbreviations ! dase antivitamin which may cause epileptic convul- l" pyridoxal 5′‑phosphate phos- sions, other severe neuronal disorders and even GABA: γ-aminobutyric acid phatase death, is also found in Ginkgo leaves and leaf-de- GAD: l" vitamin B6 rived remedies. Because of its structural similar- MPN(P): 4′-O-methylpyridoxine

ity to the B6 vitamers, an interaction of ginkgotox- (5′-phosphate), i.e., ginkgotoxin in with enzymes involved in the vitamin B6-de- (5′-phosphate) DPN(P): pendent metabolism of the human brain is possi- 4′-deoxypyridoxine (5′-phosphate) ble. This led us to investigate how the neurotoxic PKH: human pyridoxal kinase ginkgotoxin acts in the brain. To this end the gene PL(P): pyridoxal (5′-phosphate) coding for the human pyridoxine 5′-phosphate PLPP: pyridoxal 5′-phosphate phosphatase oxidase was heterologously overexpressed in E. PM(P): pyridoxamine (5′-phosphate) coli and the homogeneous enzyme was character- PN(P): pyridoxine (5′-phosphate) ized. The investigation showed that the enzyme is PNPO: pyridoxine 5′-phosphate oxidase

Introduction metabolism and the synthesis of neurotransmit- ! ters in the brain. Thus, an inhibitory or competi- Downloaded by: University of Washington at Seattle. Copyrighted material. received Dec. 18, 2008 Medications based on Ginkgo extracts are top sell- tive influence of ginkgotoxin on these reactions revised January 28, 2009 ing phytopharmaceuticals. The leaves of Ginkgo in vivo seems plausible. Indeed, it has been shown accepted February 12, 2009 biloba are the source of remedies used in the ther- for animal models that the administration of gink- ′ Bibliography apy for insufficient central and peripheral blood gotoxin and other 4 -substituted vitamin B6 ana- DOI 10.1055/s-0029-1185482 flow [1]. The characteristic ingredients which the logues (e.g., 4′-deoxypyridoxine, DPN, l" Fig. 1) Published online March 13, beneficial effects are attributed to are ginkgolides, results in an impaired amino acid metabolism of 2009 bilobalide and biflavonoids [2–5]. However, be- the brain: the production of the important inhib- Planta Med 2009; 75: 563–567 sides these, the B antivitamin ginkgotoxin (i.e., itory γ-aminobutyric acid (GA- © Georg Thieme Verlag KG 6 ′ l" Stuttgart · New York · 4 -O-methylpyridoxine, MPN) ( Fig. 1) has been BA) is significantly decreased concomitantly with ISSN 0032‑0943 found in the seeds and leaves of Ginkgo biloba, a reduction in glutamate decarboxylase activity and it is even detectable in leaf-based remedies (GAD) in different regions of the rabbit brain and Correspondence Dr. C. Drewke [6]. Ginkgotoxin is a structural analogue of the in the brain of guinea pigs [7–10]. The effects Institute of Pharmaceutical physiologically active B6 vitamins pyridoxal 5′- caused by ginkgotoxin and 4′-deoxypyridoxine Biology phosphate (PLP) and pyridoxamine 5′-phosphate were alleviated by the addition of vitamin B6 [10, University of Bonn l" Nussallee 6 (PMP) ( Fig. 2), which participate in many enzy- 11]. That is why these latter two vitamin B6 deriv- 53115 Bonn matic reactions including those of the amino acid atives are named antivitamins. Investigations on Germany rats and guinea pigs revealed that administration Phone: + 49228732563 of ginkgotoxin led to convulsions followed by Fax: + 492 28733250 * Dedicated to Dr. Heinz G. Floss on the occasion of his 75th γ [email protected] birthday. death of the animals. Also, the cerebral -amino-

Salamon N et al. Influence of Antivitamins… Planta Med 2009; 75: 563–567 564 Original Paper

Fig. 1 B6 antivitamins 4′-deoxypyridoxine (DPN), ginkgotoxin (MPN) and their 5′-phosphate esters DPNP and MPNP.

Fig. 2 Salvage pathway interconverting the B6 vitamers. PKH: human pyr- idoxal 5′-kinase, PNPO: pyridoxine (pyridoxamine) 5′-phosphate oxidase, butyric acid level was reduced with a simultaneous increase of PLPP: pyridoxal (pyridoxine, pyridoxamine) 5′-phosphate phosphatase, PN ′ ′ glutamine and glutamate concentrations [10,12]. (P): pyridoxine (5 -phosphate), PM(P): pyridoxamine (5 -phosphate), PL(P): pyridoxal (5′-phosphate). Similar symptoms had been described as the result of overcon- sumption of Ginkgo seeds in East Asia in the 1930s and 1940s [13,14]: epileptiformic convulsions, vomiting, unconsciousness and paralysis of extremities were the characteristics of this so- convulsions due to the excessive consumption of Ginkgo seeds called “gin-nan sitotoxism”. Many intoxications (up to 74%) were [17,18]. Thus, these results clearly implicate pyridoxal 5′-kinase observed in children and they were lethal in 27% of the cases as a physiological target for ginkgotoxin and provide a plausible [13]. explanation for the intoxication symptoms caused by the . Since epileptic convulsions may be the result of a glutamate/γ- However, an interaction of ginkgotoxin with further enzymes in- aminobutyric acid imbalance, gin-nan sitotoxism can well be ex- volved in the provision of active cofactor for the amino acid me- plained by inhibition of glutamate decarboxylase (GAD) [14,15], tabolism of the brain cannot be excluded. One of these is pyridox- which converts glutamate to γ-aminoburyric acid using pyridox- ine 5′-phosphate oxidase (PNPO) (EC 1.4.3.5.), another enzyme al 5′-phosphate as a cofactor. Inhibition of this enzyme by re- besides pyridoxal 5′-kinase (PKH) of the salvage pathway placement of pyridoxal 5′-phosphate by ginkgotoxin would cause (l" Fig. 2). The enzyme uses both pyridoxine 5′-phosphate and an accumulation of glutamate and thus could lead to the epilepti- pyridoxamine 5′-phosphate as substrates [19–21]. Because of formic observed during intoxication with ginkgotoxin. the structural similarity to these vitamers, an interference of However, the hypothesis that the direct inhibition of glutamate ginkgotoxin or its 5′-phosphate with pyridoxine 5′-phosphate decarboxylase by ginkgotoxin leads to an impaired amino acid oxidase seemed possible. Furthermore, it has already been

metabolism of the brain and as such triggers the symptoms of shown that 4′-deoxypyridoxine 5′-phosphate, a synthetic B6 anti- ginkgotoxin intoxication was disproven by studies of Buss et al. vitamin (l" Fig. 1), inhibits the oxidase from rabbit liver between [15]: indeed an inhibition of the glutamate decarboxylase isoen- 34% to 70.2% in high concentrations (200 µM, 1 mM) [21] de-

zyme GAD65 by ginkgotoxin 5′-phosphate was shown in vitro, but pending on the substrate (PNP or PMP) employed. A Ki value has the concentration at which this inhibition took place was physio- not been given. logically not relevant (2.7 mM). Since the highest concentration These considerations led us to investigate the antivitamins of ginkgotoxin determined in the serum of people suffering from shown in l" Fig. 1 with respect to their ability to inhibit human gin-nan sitotoxism did not exceed 6.99 µM [15–17] a significant pyridoxine 5′-phosphate oxidase in vitro. As a result, neither interaction of ginkgotoxin with glutamate decarboxylase in vivo ginkgotoxin nor its 5′-phosphate affect the activity of pyridoxine Downloaded by: University of Washington at Seattle. Copyrighted material. can be excluded. 5′-phosphate oxidase in vitro and thus very probably also not in These results prompted us to investigate the possibility that gink- vivo. We observed, however, that the enzyme is inhibited by the gotoxin interferes with the metabolism in an indi- synthetic 4′-deoxypyridoxine 5′-phosphate.

rect way through reduction of the vitamin B6 supply in the hu- man brain. A limitation of the active cofactors pyridoxal 5′-phos- phate and pyridoxamine 5′-phosphate can be caused by inhibi- Materials and Methods tion of pyridoxal 5′-kinase (PKH, i.e., human pyridoxal 5′-kinase) ! or pyridoxine 5′-phosphate oxidase (PNPO) which interconvert Subcloning of human pyridoxine 5′-phosphate oxidase " the B6 vitamers in the “salvage pathway” (l Fig. 2). Indeed, re- An EcoRI and XhoI fragment (788 bp) containing the cDNA se- cently it has been reported that ginkgotoxin is phosphorylated quence coding for human pyridoxine 5′-phosphate oxidase by recombinant human pyridoxal 5′-kinase as an alternate sub- (BC006525) was isolated from a commercially available recombi-

strate with a significantly lower Km value compared to pyridoxal nant vector (pOTB-PNPO) (Promochem GmbH; catalog no. MGC- (PL) (4.95 µM . 58.7 µM) and thus competitively inhibits the for- 953). For further expression the fragment was amplified by PCR mation of pyridoxal 5′-phosphate, the cofactor of glutamate de- along with the creation of a 5′ NdeI and a 3′ BamHI restriction site carboxylase [18]. Due to the 4′-methoxy group a better passage of the PNPO sequence using Platinium® Pfx DNA polymerase (In- ™ through the blood-brain barrier is assumed for ginkgotoxin vitrogen Life Technologies) and the primers NdeIfor:5′-ATA GGC " " (l Fig. 1) in comparison to pyridoxal (l Fig. 2). The Km value GGC CCC ATA TGA CGT GCT G-3′ and BamHIrev:5′-GCC AGC AGG determined in vitro matches the ginkgotoxin plasma levels TCC TAG GGT TAA GGT G-3′. As the direct subcloning of the se- (1.31 µM–6.99 µM) of those people suffering from epileptiformic quence into the expression vector pET-11a (Novagen) repeatedly

Salamon N et al. Influence of Antivitamins… Planta Med 2009; 75: 563–567 Original Paper 565

failed, an indirect route using a 3′-thymidine overhang vector The Km and Vmax values for PNPO were determined with the op- was chosen. For creation of this vector 10 µg of plasmid pBlue- tical test by measuring the activity of 20 µg of enzyme at increas- script® II KS(−) (Stratagene) were first linearized using restriction ing concentrations of either PNP and PMP (0.005–0.5 mM each) endonuclease EcoRV, precipitated with ethanol and resolved in as substrates.

90 µL of H2O. Subsequently 85 µL of linearized vector DNA solu- Determination of the Ki value for DPNP was performed using tion were incubated with 2 µL (100 mM) of dTTP, 6 U Taq-poly- 20 µg of pure enzyme with variable substrate concentrations merase (Qbiogene) and the respective buffer in a total volume of (0.1 to 1.5 mM) and increasing inhibitor concentrations (0.05– 100 µL for 2 h at 70°C. The sample was then extracted with an 3 mM). equal volume of Kirby mix [phenol, pH 8.0/chloroform/isoamyl (25:24 :1)]. After high speed centrifugation the DNA Synthesis of MPNP and DPNP was precipitated from the upper phase with ethanol, dried and MPNP and DPNP were synthesized by a previously described resuspended in Tris-EDTA (pH = 8) buffer. The resulting 3′-T-over- method [11], modified according to Buss et al. [15]. hang vector was ligated with the PCR-amplified PNPO sequence, which had been modified as follows: 30 µL of the gel-extracted (QIAquick Gel Extraction KIT; Qiagen) PCR product were incu- Results and Discussion bated with 0.5 µL (25 mM) of dATP, 1 U of Taq-polymerase (Qbio- ! gene) and the respective buffer in a total volume of 40 µL for Pyridoxine 5′-phosphate oxidase catalyzes the conversion of pyr- 10 min at 72°C. After propagation of the resulting recombinant idoxine 5′-phosphate and pyridoxamine 5′-phosphate to pyri- plasmid, the sequence coding for PNPO, which was proven by se- doxal 5′-phosphate [19–21] (l" Fig. 2). The sequence of the gene quencing to be free from any mismatches, was isolated using re- encoding the human pyridoxine 5′-phosphate oxidase striction endonucleases NdeI and BamHI and inserted into the re- (BC006525) heterologously expressed in E. coli K12 DH10B was spective restriction sites of vector pET-11a (Novagen). After a fur- identical to that found in the common databases [National Center ther propagation step in E. coli XL1 Blue MRF′ (Stratagene) the re- for Biotechnology Information (NCBI)]. The molecular weight combinant plasmid pET-11a-PNPO was transferred to the ex- (30 kDa, SDS PAGE) of the encoded protein is in agreement with pression host E. coli BL21(DE3) (Stratagene). This system allowed the expected calculated mass. The two-step purification proce- the heterologous expression of the gene coding for human PNPO dure of enzmye from 1 L of recombinant bacterial culture re- as a native untagged protein. sulted in a 4.9-fold enzyme enrichment yielding 1.43 mg of ho- mogeneous protein with an activity of 49.1 µmol/mg·min−1. Expression, purification, and enzymatic assays of PNPO The enzymatic activity was stable at − 20 °C for 12 days, and de- The recombinant strain BL21 (DE3) (pET-11a-PNPO) was grown creased to 50% over a period of 97 days. For kinetic measure- in LB medium containing penicillin G (100 µg/mL) at 37°C to an ments the storage period of the enzyme did not exceed 12 days. optical density (OD600) of 0.5. Isopropyl thio-β-D-galactoside Cofactor of the human pyridoxine 5′-phosphate oxidase is FMN (IPTG) was added to a final concentration of 0.4 mM, and the cul- [19–21], which appeared to be bound tightly to the enzyme, as ture was incubated with shaking for further 24 hours and har- the solution of the purified protein exhibited a yellowish color vested by centrifugation at 4000 g for 20 min. Expression of the and showed enzymatic activity without extra addition of FMN. protein was controlled by SDS-PAGE [22]. The cell pellet was re- Maximum activity was observed for a pH range of 8.0–9.0. All as- suspended in 10 mL of lysis buffer (50 mM NaH2PO4, 300 mM says were performed at pH 8.4. The velocity of the reaction NaCl, 10 mM imidazole, pH 8.0) at 10 mL per 1 L of culture and showed a clear optimum at 46°C. However, to create physiologi- frozen over night. After thawing, the cells were subjected to ul- cal conditions for determination of the effect of antivitamins on trasonic treatment (Branson sonifier; 20 times, 15 s with 15 s in- the human enzyme, measurements were carried out at 37°C. tervals, 50% output at stage 5) under permanent cooling on ice. The kinetic data for pyridoxine 5′-phosphate oxidase with the The crude extract (from 6 L culture) derived from ultrasonic two substrates are given in l" Table 1. treatment was subsequently subjected to affinity chromatogra- Both unphosphorylated antivitamins ginkgotoxin and 4′-deoxy- Downloaded by: University of Washington at Seattle. Copyrighted material. phy and gel filtration according to Cash et al. [23] as modified by pyridoxine did not show any effect on the enzyme up to a con- Kästner et al. [18]. In this procedure, the cell free extract was centration of 2 mM and 5 mM, respectively. This was expected, passed over EAH-Sepharose™ 4B (Amersham Biosciences Europe since the pyridoxine 5′-phosphate oxidase does not accept un-

GmbH) coupled to pyridoxine phosphate. After washing of the phosphorylated B6-vitamers [25]. The same observation was column, the oxidase was eluted using a buffer containing 50 mM made for 5′-phosphorylated ginkgotoxin (l" Fig. 3A). As a control of pyridoxine phosphate and, in order to remove the pyridoxine 4′-deoxypyridoxine 5′-phosphate, the synthetic B6 antivitamin, phosphate containing buffer, subsequently was subjected to gel was also tested and clearly found to inhibit the enzyme filtration. (l" Fig. 3B). The inhibition was alleviated by increasing amounts The procedure resulted in homogeneous protein as proven by de- of pyridoxine 5′-phosphate (l" Fig. 3C) indicating a competitive tection of a single band in a silver stained SDS gel (data not inhibition. A Ki value for 4′-deoxypyridoxine 5′-phosphate of shown). Determination of PNPO activity was performed in Tris- 0.42 × 10−3 M (Hanes-Woolf) was calculated (l" Table 1). The fea- HCl buffer (0.1 M, pH 8.4) in the presence of FMN at 414 nm mon- ture of 4′-deoxypyridoxine 5′-phosphate to be a potent inhibitor itoring the increase of absorption based on a Schiffʼs base forma- of pyridoxine 5′-phosphate oxidase from rabbit liver has already −3 tion between Tris and PLP according to the method of Bahn et al. been reported by Wada and Snell [21]. A Ki value (0.11 × 10 M), [24]. A standard assay was performed for 10–40 min at 37°C in a however, was only published for the bacterial enzyme [26]. This total volume of 100 µL containing 2 µg/µL of purified enzyme and is only four times lower compared to the value we determined for 0.002 mM FMN. The reaction was started by addition of 10 µL of our human oxidase (l" Table 1). PNP (5 mM). Results of enzymatic assays are the mean of two dif- The results show that ginkgotoxin or its 5′-phosphate have no in- ferent experiments with three individual measurements each. fluence on the human pyridoxine 5′-phosphate oxidase in vitro.

Salamon N et al. Influence of Antivitamins… Planta Med 2009; 75: 563–567 566 Original Paper

Table 1 Kinetic data of pyridoxine 5′-phosphate oxidase (PNPO) with B6 vitamers PNP and PMP and inhibition constant [Ki (M)] of the PNPO inhibitor DPNP. No inhibition of PNPO was observed for a ginkgotoxin 5′-phosphate (MPNP) concentration up to 2 × 10−3 M.

−1 −1 −1 −1 Compound Km [M] Vmax [mol/mg × min ]Kcat [s ]Kcat/Km [mol ×s ]Ki [M] Pyridoxine 5′-phosphate (PNP) 5.90 ± 0.48 × 10−6 4.33 ± 0.53 × 10−7 0.22 3.68 × 104 – Pyridoxamine 5′-phosphate (PMP) 1.49 ± 0.15 × 10−5 1.57 ± 0.36 × 10−7 0.08 5.30 × 104 – 4′-Deoxypyridoxine 5′-phosphate –– –– 0.42 × 10−3 (DPNP)

Fig. 4 Hypothesis summarizing metabolic steps of ginkgotoxin (MPN),

ginkgotoxin 5′-phosphate (MPNP) and B6 vitamers (PN: pyridoxine; PM: pyridoxamine; PL: pyridoxal) and their 5′-phosphates PNP, PMP and PLP as catalyzed by human pyridoxal kinase (PKH), pyridoxal 5′-phosphate phos-

phatase (PLPP) and glutamate decarboxylases GAD65 and/or GAD67.

This parallels preliminary observations we made (data not shown) for the human pyridoxal 5′-phosphate phosphatase which after overexpression and incubation in a crude enzyme ex- tract was unaffected by the presence of the same antivitamins. This leaves the human pyridoxal 5′-kinase as the only tested en- zyme that is severely affected by ginkgotoxin due to the ex- Downloaded by: University of Washington at Seattle. Copyrighted material. −6 tremely low Km value observed for ginkgotoxin (4.95 × 10 M) when compared to its natural substrate pyridoxal (5.87 × 10−5 M) [18]. Our current knowledge on the influence of ginkgotoxin and its 5′-

phosphate on vitamin B6 metabolism can therefore be summar- ized as shown in l" Fig. 4. After intestinal absorption of vitamin B6 and ginkgotoxin these metabolites are distributed by the blood stream and phosphorylated (A) (compare l" Fig. 2). Phos- phorylated B6 vitamers (PNP, PMP, PLP) and ginkgotoxin 5′-phos- Fig. 3 Reversible inhibition of pyridoxine 5′-phosphate oxidase (PNPO) by phate are hydrolyzed by pyridoxal 5′-phosphate phosphatase (B) 4′-deoxypyridoxine 5′-phosphate (DPNP), but not by ginkgotoxin 5′-phos- [27]. Due to its relative lipophilicity this is likely to be followed by phate (MPNP) during formation of pyridoxal 5′-phosphate (PLP). A 100% a preferential uptake of ginkgotoxin through the blood/brain bar- PNPO activity in the presence of MPNP (0.5 mM, 2 mM). B Inhibition of rier (C). As long as ginkgotoxin is present in the brain the human PNPO activity by increasing amounts of DPNP (0.05 to 3.0 mM) in the pyridoxal 5′-kinase will preferentially rephosphorylate ginkgo- presence of pyridoxine 5′-phosphate (PNP, 0.05 mM) as a substrate. C Re- version of DPNP (0.5 mM) caused inhibition by increasing amounts of PNP toxin rather than vitamin B6 (D) which in turn may lead to a re- (0.1 to 1.5 mM). Results are the mean of two different experiments with duced level of B6 vitamins in the brain, an impaired activity of three individual measurements each. glutamate decarboxylase (E) and a surplus of glutamic acid over γ-aminobutyric acid [18]. This in turn may precipitate seizures and other neuronal disorders.

Salamon N et al. Influence of Antivitamins… Planta Med 2009; 75: 563–567 Original Paper 567

uration and the preictal activation of glutamate decarboxylase activity. With glutamate decarboxylases GAD65 and GAD67 [15], pyridoxal – kinase (PKH) [18], pyridoxal 5′-phosphate phosphatase (PLPP) J Neurochem 1980; 34: 822 830 ′ 9 Haug P, Nitsch C. Increase in taurine content before onset of seizures in- and pyridoxine 5 -phosphate oxidase (PNPO) (this publication) duced by a glutamate decarboxylase inhibitor. Exp Brain Res 1982; 48: the essential human enzymes for being most probable targets 463–466 for ginkgotoxin or its 5′-phosphate have been investigated in vi- 10 Wada K, Haga M. Food poisoning by Ginkgo biloba seeds. In: Hori T, tro with respect to their interaction with ginkgotoxin and its 5′- Ridge RW, Tulecke W, Del Tredici P, Tremouillaux-Guiller J, Tobe H, edi- tors. Ginkgo biloba – a global treasure, from biology to medicine. 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Salamon N et al. Influence of Antivitamins… Planta Med 2009; 75: 563–567