Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5¢-phosphate oxidase Jeong Han Kang 1 , Mi-Lim Hong 1 , Dae Won Kim 2 , Jinseu Park 2 , Tae-Cheon Kang 3 , Moo Ho Won 3 , Nam-In Baek 4 , Byung Jo Moon 1 , Soo Young Choi 2 and Oh-Shin Kwon 1 1 Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu, Korea; 2 Department of Genetic Engineering, Division of Life Sciences, and 3 Department of Anatomy, College of Medicine, Hallym University, Chunchon, Korea; 4 Graduate School of Biotechnology & Plant Metabolism Research Center, Kyunghee University, Suwon, Korea We used a combined computer and biochemical approach to characterize human pyridoxine 5¢-phosphate oxidase (PNPO). The human PNPO gene is composed of seven exons and six introns, and spans approximately 8 kb. All exon/intron junctions contain the gt/ag consensus splicing site. The absence of TATA-like sequences, the presence of Sp1-binding sites and more importantly, the presence of CpG islands in the regulatory region of the PNPO gene are characteristic features of housekeeping genes. Northern blot analyses showed two species of poly(A) + RNA of  2.4 and  3.4 kb at identical intensity, whereas Western blot analysis showed that no protein isoform exists in any of the tissues examined. PCR-based analysis led to the idea that two messages are transcribed from a single copy gene, and that the size difference is due to differential usage of the poly- adenylation signal. The major sites of PNPO expression are liver, skeletal muscle and kidneys while a very weak signal was detected in lung. The mRNA master dot-blot for mul- tiple human tissues provided a complete map of the tissue distribution not only for PNPO but also for pyridoxal kinase and pyridoxal phosphatase. The data indicate that mRNA expression of all three enzymes essential for vitamin B 6 metabolism is ubiquitous but is highly regulated at the level of transcription in a tissue-specific manner. In addition, human brain PNPO cDNA was expressed in Escherichia coli, and the roles of both the N- and C-terminal regions were studied by creating sequential truncation mutants. Our results showed that deletion of the N-terminal 56 residues affects neither the binding of coenzyme nor catalytic activity. Keywords: deletion mutation; genomic organization; PNP oxidase; polyadenylation; tissue distribution. Pyridoxal 5¢-phosphate (PLP), the metabolically active form of vitamin B 6 , is a required coenzyme for numerous enzymes involved in amino acid metabolism [1]. The functions of PLP include coenzymatic participation in reactions leading to the formation of several neurotrans- mitters [2]. Moreover, it appears that PLP modulates steroid–receptor interactions and is involved in the regula- tion of immune function [3]. The enzymes that are conventionally involved in vitamin B 6 metabolism are an ATP-dependent pyridoxal kinase (PDXK; EC 2.7.1.35) [4,5], FMN-dependent pyridoxine 5¢-phosphate oxidase (PNPO, EC 1.4.3.5) [6,7] and pyridoxal phosphatase (PDXP, EC 3.1.3) [8,9]. PNPO catalyzes the conversion of pyridoxine 5¢-phos- phate (PNP) and pyridoxamine-5¢-phosphate (PMP) to PLP, with O 2 as an electron acceptor. Kinetic studies published by Choi et al. [10], have established that the oxidase can function via either a binary or ternary complex mechanism, depending upon the nature of the substrate. The enzyme isolated from mammalian tissues is a dimer composed of two identical subunits each of  30 kDa. FMN acts as a coenzyme and is absolutely required for catalytic activity [11]. Extensive studies with the Escherichia coli enzyme revealed that there are two molecules of FMN per dimer and not one FMN as reported previously [12]. The enzyme was first obtained in pure from rabbit liver and several of its properties were characterized [13]. It has also been studied in preparations from pig brain [14], sheep brain [15], yeast [16], and bacteria [17–19]. Interestingly, Ngo et al. [7] reported that no PNPO activity was detected in liver and neurally derived tumour cells, which suggested that tumour tissue uses a different pathway for the synthesis of PLP than that used by normal tissues. Thus the absence of oxidase activity and its relationship to other metabolic processes occurring in abnormal cells remains to be explained. The characterization of the cDNA encoding PNPO opens new avenues of research designed to under- Correspondence to O S. Kwon, Department of Biochemistry, Kyungpook National University, Taegu, 702-701, Korea. Fax: + 82 53 943 2762, Tel.: + 82 53 950 6356, E-mail: oskwon@knu.ac.kr and S.Y. Choi, Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon, 200-702, Korea. Fax: + 82 33 241 1463, Tel.: + 82 33 248 2112, E-mail: sychoi@hallym.ac.kr Abbreviations: PLP, pyridoxal 5¢-phosphate; PNPO, pyridoxine 5¢-phosphate oxidase; PNP, pyridoxine 5¢-phosphate; PMP, pyridox- amine 5¢-phosphate; PDXK, pyridoxal kinase; PDXP, pyridoxal phosphatase; EST, expressed sequence tag; EBI, European Bioinformatics Institute. Enzymes: ATP-dependent pyridoxal kinase (EC 2.7.1.35); FMN- dependent pyridoxine 5¢-phosphate oxidase (PNPO, EC 1.4.3.5); pyridoxal phosphatase (EC 3.1.3). (Received 23 February 2004, revised 16 April 2004, accepted 20 April 2004) Eur. J. Biochem. 271, 2452–2461 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04175.x stand the structure and regulatory mechanisms of this enzyme. A high degree of sequence homology exists between PNPO from different sources suggesting that all members of this enzyme group share a common three-dimensional fold and catalytic mechanism. Recently, the E. coli [20–22] and human enzymes [23] have been cloned and crystallized. In contrast with the abundant data on the mechanism of catalysis very little is known about the genomic structure and expression of PNPO. Here we present a characteriza- tion of the genomic organization, the structure of the mRNA isoforms produced by alternative polyadenylation, and the tissue distribution of the transcript. To our knowledge this study describes the first detailed investiga- tion of the transcription of human PNPO. In addition, the minimum size necessary for enzymic function was deter- mined by deletion mutagenesis. Materials and methods Materials A Marathon-Ready TM cDNA library from human brain, a multiple tissue Northern blot (MTN TM Blot) and a dot blot array (MTE TM Array) containing poly(A) + RNAs from human tissues were purchased from Clontech. pET-28a(+) expression vector from Novagen, and restriction endonuc- leases and other cloning reagents were from New England Biolabs Inc. or Promega. Double-stranded DNA probes were radiolabeled with [a- 32 P]dCTP (3000 CiÆmmol )1 )using a commercial random priming kit (both from Amersham Pharmacia Biotech). Human tissue specimens for Western blot analysis were obtained from The Medical Center, Hallym University, Chunchon, South Korea, and approved by the Institutional Review Board. Cloning and deletion mutagenesis NCBI BLAST searches revealed an expressed sequence tag (EST) clone (GenBank TM accession number AK001397) encoding a full-length ORF for human PNPO. This clone was used to design PCR primers for the cloning of human brain PNPO gene. We used a PCR amplification using wild- type PNPO specific primers (Table 1) and Marathon Ready cDNA library (human whole brain, Clontech) as a template. PCR was carried out in GeneAmp PCR system 2400 (PerkinElmer Life Sciences) for 30 cycles of denaturation 94 °C for 1 min, annealing 55 °C for 1 min and extension 72 °C for 2 min. The PCR product was cloned into the pGEM-T vector (Promega) and sequenced (GenBank TM / EBI accession number AF468030). To facilitate expression vector construction, a BamHI recognition site was introduced at both ends of the ORF by PCRwithprimersshowninTable1.ThePCRmixturewas analysed on a 0.8% agarose gel, and the product band was extracted from the gel, purified, and ligated into the pGEM vector. Then a BamHI digested fragment was subcloned into pET28a expression vector (pET28a/PNPOx) and used to transform BL21(DE3) competent cells. For the construction of deletion mutants, convenient restriction sites and PCR-based strategies were used (Table 1). Each PNPO deletion mutant was subcloned into pET28a. These constructs encode the following residues of human PNPO: D1–56, residues 57–262; D1–72, residues 73–262; D238–262, residues 1–237. The structures of these plasmids were verified by restriction and sequence analysis to ensure that the reading frame was maintained. In silico analysis The full-length ORF sequence of PNPO (GenBank TM /EBI accession number AF468030) was used to query human genome sequences using BLASTN in order to elucidate the genomic structure. To identify putative transcription factors binding sites in the promoter regions, an analysis of the 5¢-upstream sequence of the PNPO gene was performed in silico by using the MATINSPECTOR PROFESSIONAL program in genomatix suite (http://www.genomatix.de) [24] and TFSEARCH software (http://www.cbrc.jp/research/db/ TFSEARCH.html). The CpG island as defined by Gardiner- Garden and Frommer [25] was analysed using CpG plot/ CpG report [26] of the European Molecular Biology Open Software Suite (EMBOSS). The program CPGPLOT was used to plot all CpG rich areas. Northern analysis A Northern filter containing eight human tissue-specific poly(A) + RNAs and a dot blot array containing human poly(A) + RNAs from various adult tissues, foetal tissues, and cancer cell lines were prehybridized at 65 °Cfor1h in ExpressHyb TM Hybridization solution (Clontech). The filters were then hybridized at 65 °Cfor16hwith 32 P-labelled specific cDNA probes containing either the complete ORF or the 3¢-UTR of PNPO as required. The 3¢-UTR of 1 kb had been cloned using the PNPO-specific primers (sense, 5¢-TACACAGGGTGGTCCACAAGC Table 1. PCR primers used in the expression constructions for wild-type and deletion mutants. PNPO deletion mutants were constructed using PCR amplication of the relevant portions of PNPO cDNA followed by restriction digestion and subsequent subcloning into pGEM and pET28a vector. Primer Primer sequence Restriction enzyme Wild Forward 5¢-TAAGGATCCCCCATGACGTGC-3¢ BamHI Reverse 5¢-CAGGATCCAGAGTTAAGGTGCAAG-3¢ BamHI D1–56 Forward 5¢-CCGAATTCGACCCAGTGAAACAGTTT-3¢ EcoRI Reverse 5¢-GGAAGCTTAGTTAAGGTGCAAGTCTCTC-3¢ HindIII D1–72 a Forward 5¢-CGGATCCGAGGAGGCTGTTCAGTGT BamHI D238–262 a Reverse 5¢-AGGATCCCTAGGGTAGGCCCCGCCG-3¢ BamHI a The reverse and forward primer of wild-type were used for constructions of D1–72 and D238–262, respectively. Ó FEBS 2004 Human pyridoxine 5¢-phosphate oxidase (Eur. J. Biochem. 271) 2453 CAGG-3¢;antisense,5¢-GGGGCGGTAACGGCTGG ACAGAGAA-3¢). To obtain the full-length ORF, we performed PCR amplifications using the specific primers for human PDXP [9] and human PDXK (sense, 5¢-CAG GCCCCATATGGAGGAGGAGTGCCGG-3¢;antisense, 5¢-GGGGATCCTCACAGCACCGTGGC-3¢) [27]. After washing as recommended by the manufacturer, blots were exposed to X-ray films at )70 °C with an intensifying screen for the appropriate time period. Blots were reprobed with a human b-actin as a loading control. For scanning densi- tometry, the blot was scanned and BioLab Image software was used to quantify the signals. Western analysis The proteins separated by SDS/PAGE were electropho- retically transferred to nitrocellulose membrane, and the membrane was rinsed briefly in distilled water and then air-dried. The blot was blocked with Blotto (Bio-Rad, Richmond, VA, USA) for 1 h at 37 °C. After rinsing with TBS, the blots were incubated for 1 h with a mAb against sheep PNPO [28], then washed three times in TBS containing Tween 20 at 5 min intervals. The membrane was incubated for 1 h at 37 °C with horseradish peroxidase- conjugated, goat antimouse IgG antibodies, and diluted 1 : 5000 in TBS containing 0.05% (v/v) Tween-20. Finally, the bound conjugate was identified by incubating the membrane in a substrate buffer [0.5 mgÆmL )1 4-chloro-1- naphtol in 1 : 5 (v/v) methanol/TBS and 0.015 H 2 O 2 ]for 5 min at room temperature. Expression in E. coli and purification of recombinant human PNPO The PNPO cDNA was cloned between the BamHI of pET28a expression vector (Novagen Inc.) after PCR amplification. Transformants of E. coli BL21(DE3) har- bouring pET28a/PNPO were cultured at 37 °CinLuria– Bertani medium with 50 lgÆmL )1 kanamycin. When that culture had grown to an A 600 of 0.5, isopropyl thio-b- D -galactoside was added to a final concentration of 1 m M . After inducing the expression of the PNPO protein for 3 h at 37 °C, cells were harvested by centrifugation (10 000 g at 4 °C for 10 min), and the pellet was suspended in lysis buffer (20 m M Tris/HCl pH 7.4, 1 m M EDTA, 200 m M NaCl, 10 m M 2-mercaptoethanol, 0.5 m M phenyl- methylsulfonyl fluoride). The cell suspension was sonicated, and the lysate was cleared by centrifugation at 12 000 g and 4 °C for 20 min. The supernatant was then poured into the column loaded with nickel-nitrilotriacetic acid agarose (Qiagen), washed with Tris buffer containing 40 m M imidazole, and protein was eluted with 200 m M imidazole. The purity of the eluted protein was evaluated by SDS/ PAGE on 12% acrylamide and visualized using Coomassie blue staining. Enzyme assay The spectrophotometric method was used in the assay of PNPO activity. The rate of the formation of PLP was measured by following the increase in absorbance at 410 nm in 0.1 M Tris/HCl pH 8.4 containing 0.1 m M PNP. At this wavelength, the Schiff base formed between Tris and PLP has an extinction coefficient of 5900 M )1 Æcm )1 . One unit of specific activity is defined as the amount of protein that catalyses the formation of 1 lmol PLPÆmin )1 at 25 °C. The value of K m and k cat were determined from double reciprocal plots of initial velocity and substrate concentra- tion. The concentration of enzyme was determined by the Bradford method. Results and discussion Genomic organization of human PNPO Using PNPO cDNA as a query sequence, a BLAST analysis (available through the NCBI web site) mapped the PNPO gene to human chromosome 17q21.32. The gene spans over 7743 bp, and the coding region of the gene was divided into seven discrete exons as shown in Fig. 1A. All exon/intron boundaries were found to contain the canonical 5¢ donor GT and 3¢ acceptor AG sequences (Table 2). The ORF encodes a 261 amino acid protein with a molecular mass of 30 kDa. A computer calculation reveals that the isoelectric point for the protein is 6.61. SCANPROSITE software analysis by EXPASY showed that the deduced human protein has the following putative post-translational modification sites: a sulfation site, nine phosphorylation sites, three N-myristoylation sites and an RGD cell attachment sequence. The genomic sequences were examined for the presence of CpG islands using the CpG plot program from the European Bioinfor- matics Institute (EBI). The human PNPO gene contains CpG islands with a CG obs /CG exp ratioinexcessof0.6anda G + C content of 62% spanning two regions from )377 to )158 and from )137 to +136 of the start codon. Such a CpG island is indicative of the presence of a promoter region and indicates a widespread expression. Analysis of the 5¢-flanking human PNPO gene sequence using PROMOTOR- INSPECTOR software (Genomatix Software GmbH, Munich, Germany) resulted in no apparent core promoter region. The MATINSPECTOR program in Genomatix, however, Fig. 1. Genomic organization of the PNPO. Schematic diagram of the exon/intron organization of the human (A) and mouse (B) PNPO gene. Exons are designated by closed boxes, and introns by bold lines. The ORF is marked black, and grey boxes denote the 5¢-and3¢-UTR sequences. The locations of CpG islands are indexed relative to the start codon, and indicated by the open boxes with numbers. 2454 J. H. Kang et al. (Eur. J. Biochem. 271) Ó FEBS 2004 revealed that ) similar to the mouse ) the proximal 5¢-flanking region lacked a TATA-box but contained two Sp1 sites (data not shown). The absence of a TATA-box is indeed a noticeable feature of many housekeeping genes [29]. The mouse gene encodes a protein of 261 amino acids of m 30 114 Da, and it is located on chromosome 11 which has a very similar genomic organization to that of humans (Fig. 1B). The longest cDNA contains 1991 bp consisting of a 786 bp ORF, a 118 bp 5¢-untranslated region and a 1087 bp 3¢-noncoding region. As in humans, the mouse PNPO gene is encoded by seven exons and the intron/exon junctions also follow the GT/AG rule. The 3¢-end of the sequence contains a poly(A) stretch, preceded by a putative polyadenylation signal AATAAA. The mouse PNPO gene has CpG islands extending from position )511 to 276 and from )82 to +227 with a CG content of 61%. The deduced protein with a predicted pI of 8.35 has a putative sulfate site, eight phosphorylation sites, two N-myristoylation sites and one RGD cell attachment sequence. Human and mouse PNPO share 90% identity at the amino acid level. Table 2. The intron/exon junctions of the human PNPO gene. The nucleotide sequences at exon (uppercase letters) and intron (lowercase letters) junction are shown. Exon and intron sizes are indicated in bp. Exon (bp) 5¢-splice donor Intron (bp) 3¢-Splicing acceptor Exon I (243) CGAGAG/gtgccg 1 (1492) tcctag/GCATTT II II (125) CACCAG/gtgggc 2 (1185) tcctag/AGATGG III III (100) GAGCTG/gtgggt 3 (843) ttctag/GACTCT IV IV (54) CGTCAG/gtgagt 4 (248) gagcag/GTGCGT V V (129) CGGGAG/gtgagt 5 (333) ggacag/TATCTG VI VI (71) ATCCTG/gtgagt 6 (220) ttatag/GGGTGG VII VIIa (1662) AGATTA VIIb (2700) ATTGAT Consensus G/gtg ag/ Fig. 2. Splicing pattern of the PNPO mRNA isoforms. (A) Northern blot analysis of the expression of the PNPO gene in human tissues. Two micrograms of poly(A) + RNA prepared from the tissues indicated were analysed by Northern hybridization. The blots in the upper panel were hybridized with 32 P-labelled probes corresponding to the coding region (left) and the 3¢-UTR of human PNPO cDNA (right). The membrane was stripped and reprobed with a b-actin cDNA probe (bottom). The approximate sizes of the isoforms are indicated. (B) The scheme of two mRNA species is given. Exons are indicated by open boxes, and coding regions and UTR used for probes are delineated by black and grey box, respectively. The putative polyadenylation signal is indicated. Ó FEBS 2004 Human pyridoxine 5¢-phosphate oxidase (Eur. J. Biochem. 271) 2455 Northern blot analysis of human PNPO To determine the size of human PNPO mRNA transcripts, Northern blot analyses were performed with the full-length PNPO cDNA. As shown in Fig. 2, the PNPO mRNAs are expressed in all human tissue examined, but their relative abundance varies markedly. Of note, two transcripts of 2.4 and 3.4 kb were detectable with almost identical intensity in all tissues examined (Fig. 2A, left). Although performed under very stringent conditions, all blots revealed the presence of double bands. BLAST analysis suggests that both signals arise from the PNPO locus as there were no data to indicate the existence of a highly related gene that cross-hybridizes with the PNPO probe. There are several possible mechanisms by which multiple transcripts could be generated from the same gene: (1) use of alternative polyadenylation sites; (2) use of alternate transcription start sites; and (3) differential splicing of pre-mRNA. In the Western blot analysis as shown in Fig. 3, no protein with a molecular mass higher than 30 kDa could be detected with mAbs against sheep PNPO. This line of evidence may rule out the existence of an alternative splicing product. To further elucidate the presence of isoform message, this filter was reprobed with the DNA probes specific for the 3¢-UTR between the two potential poly(A) signals. The results showed that only the 3.4 kb band was detected (Fig. 2A, right), which supports the hypothesis that the two mRNA species are generated by alternate usage of poly- adenylation sequences. The putative schematic structure of the mRNA isoforms is shown in Fig. 2B. Two putative polyadenylation signals ) one an ATT AAA motif 1472 bp downstream of the termination codon and the other an AATAAA motif 27 bp upstream of the end of the gene ) were found within the genomic primary sequence. It is known that the most common polyadeny- lation signal is AATAAA, and that ATTAAA is  80% as efficient as the terminal sequence [30]. Thus, both polyade- nylation sites of PNPO worked, implying some read- through of the first site by an unknown mechanism. A search of the human EST database with the human PNPO sequence also supported this hypothesis. Alternate usage of polyadenylation signals is frequently seen in testis tissue. However, in mouse, such putative isoforms resulting from the alternative usage of polyadenylation could not be found in EST sequences. Human cells, unlike cells of other mammalian species, generate more than one PNPO tran- script, resulting from the preferential poly(A) site selection. This feature strongly suggests the possibility of evolutionary changes of the 3¢-UTR, which is characterized by more degrees of freedom than the 5¢-UTR and the ORF [31,32]. Tissue distribution of PNPO, PDXK and PDXP As shown in Fig. 2, Northern blot analysis indicated that the mRNA level of PNPO is highest in liver. Skeletal muscle and kidney contained considerable amounts of the tran- script while lower levels were detected in lungs. In addition, a human multiple tissue expression array (MTE TM )was analysed by hybridization with mRNAs from various human tissue. As shown in Fig. 4, we provide a complete set of the tissue distribution of PNPO mRNA in humans. Although the level of mRNA expression in the brain is low compared to that in other organs such as the liver, a densitometric analysis of the dot blot array showed a similar basal expression of PNPO in the entire brain subregion. The transcripts of foetal PNPO are relatively low compared with those of adults. Notably, the widespread distribution of PNPO in human tissue is consistent with its essential role in cellular metabolism. Another interesting aspect of our work is the finding that three key PLP metabolic enzymes, PNPO, PDXK and PDXP have remarkably different expression profiles. The Fig. 3. Western blot analysis of human PNPO. SDS/PAGE (A) and immunoblot with mAb (B) for human tissue and cell homogenates. LaneM,Molecularmassstandards;lane1,brain;lane2,liver;lane3, lung; lane 4, prostate; lane 5, human breast cancer (MCF-7); lane 6, human uterine carcinoma (HL3T1); lane 7, stomach tissue. 2456 J. H. Kang et al. (Eur. J. Biochem. 271) Ó FEBS 2004 mRNA expression levels in selected tissue for each enzyme are shown in Table 3. Consistent with their ubiquitous role in vitamin B 6 metabolism, all three transcripts have been detected in a wide variety of tissue. Analysis of the array revealed that human PDXK was expressed in essentially all organs with the highest levels observed in descending order testes, kidneys and placenta. A relatively high level of PDXK transcript was expressed in foetal organs. In contrast, human PDXP mRNAs appear to be strikingly abundant in the brain indicating a more specific role [9]. These results imply that the three enzymes are differentially expressed and regulated in a tissue specific manner. The regulation of PLP could be controlled by several factors. The synthesis of PLP requires the joint action of PDXK and PNPO, and the PLP availability is dependent on the degree of protein binding of the synthesized coenzyme and transport of the precursors [33,34], and phosphatase action [35]. PNPO does play a kinetic role in regulating in vivo PLP formation [2,36], whereas PDXK plays an additional trapping role whereby pyridoxal is diffusible across the cell membrane [33]. Tissue with high oxidase activities, however, produce PLP not only for internal consumption, but also for an external supply to other tissue with low oxidase activities. Thus, the complete metabolic network for PLP homeostasis remains to be investigated. Functional organization by deletion mutagenesis To investigate enzymatic properties, cDNA-encoded human PNPO was expressed in E. coli as a fusion protein with a His tag. The size of the recombinant protein, as well Fig. 4. Multiple tissue analysis of human PNPO mRNA expression. Tissue-specific expression of the PNPO mRNA was analysed with poly(A) + RNA dot-blot. The human multiple tissue expression (MTE TM ) array was hybridized with a 32 P-labelled PNPO-specific cDNA probe. Tissue sources for the RNA are indicated below the blot. Ó FEBS 2004 Human pyridoxine 5¢-phosphate oxidase (Eur. J. Biochem. 271) 2457 as the purity, was determined by SDS/PAGE. As shown in Fig. 5A, the fusion protein of a wild-type PNPO showed an apparent molecular mass of 34 kDa, in good agreement with the theoretical size (33.5 kDa). Recombinant PNPO was catalytically active. Steady-state kinetic analyses were carried out on the recombinant enzyme. The apparent K m of 2.1 l M and 6.2 l M were obtained for the substrate PNP and PMP, respectively, from Lineweaver–Burk (double-reciprocal) plots (Table 4). In order to delineate the region of human PNPO that is essential for catalysis, we expressed the sequential trunca- tion mutants in E. coli and determined the effect of each deletion on activity. In this work, the role of both the N- and C-terminal regions of human PNPO were studied by the truncation mutants: D1–56, D1–72 and D238–262 (Fig. 5B). V max values of 0.10 and 0.05 lmolÆmin )1 Æmg )1 for the recombinant wild-type enzyme were obtained for PNP and PMP, respectively, whereas the deletion of the noncon- served 56-amino acid at N-terminal domain (D1–56) caused about a twofold increase in catalytic activity (Table 4). The K m value of the mutant, however, is about threefold higher Table 3. Comparison of mRNA expression levels of vitamin B 6 regula- ting enzymes. A dot blot array containing human poly(A) + RNAs from various tissues were hybridized with probes as described in Fig. 4. Expression levels of selected tissues for PNPO, PDXK, and PDXP are compared. Values are given relative to the highest expressing tissue for each enzyme that was arbitrarily set to 100. PNPO PDXK PDXP Whole brain 23.8 29.0 83.6 Cerebral cortex 25.9 27.0 100.0 Frontal lobe 12.4 14.3 81.8 Parietal lobe 13.6 32.2 82.4 Occipital lobe 14.7 25.3 89.1 Temporal lobe 12.6 24.4 84.6 Paracentral gyrus of cerebral cortex 9.5 18.5 73.2 Pons 6.2 10.3 47.9 Cerebellum, left 13.3 21.9 90.5 Cerebellum, right 25.0 31.1 91.1 Corpus callosum 18.4 19.8 37.7 Amygdala 13.0 22.6 94.2 Caudate nucleus 19.0 28.4 74.2 Hippocampus 12.3 24.6 81.8 Medulla oblongate 5.0 15.8 41.7 Putamen 2.0 17.9 54.2 Accumbens nucleus 5.2 19.4 70.8 Thalamus 10.8 23.1 85.2 Spinal cord 1.6 3.7 10.8 Heart 3.7 9.1 18.7 Aorta 1.2 4.4 0.1 Atrium, left 14.0 14.7 19.6 Atrium, right 11.6 14.7 25.0 Ventricle, left 4.1 16.7 17.2 Ventricle, right 20.5 21.7 21.7 Interventricular septum 16.2 34.8 39.9 Apex of the heart 1.8 18.2 34.4 Oesophagus 7.5 13.4 2.8 Stomach 17.2 43.5 26.1 Duodenum 9.3 27.2 18.9 Jejunum 13.3 43.1 26.1 Ileum 5.8 23.0 21.2 Ilocecum 6.1 60.6 35.9 Appendix 0.7 20.5 21.2 Colon, ascending 1.2 5.7 21.9 Colon, transverse 7.9 8.0 21.6 Colon, desending 2.8 9.4 6.5 Rectum 2.3 16.4 15.8 Kidney 85.6 58.0 31.4 Skeletal muscle 34.1 15.4 20.7 Spleen 13.5 32.0 8.8 Thymus 6.4 29.9 18.6 Peripheral blood leukocyte 0.4 26.3 13.3 Lymph node 4.6 51.2 17.8 Bone morrow 11.2 42.0 22.8 Trachea 4.8 23.0 5.8 Lung 4.6 24.8 7.6 Placenta 30.2 61.4 8.1 Bladder 6.8 12.4 9.2 Uterus 2.9 19.4 9.5 Prostate 16.5 37.0 18.2 Testis 8.4 100.0 49.3 Ovary 4.3 20.1 28.9 Liver 100.0 56.0 61.8 Table 3. (Continued). PNPO PDXK PDXP Pancreas 2.5 59.0 33.3 Adrenal gland 11.5 34.4 32.6 Thyroid gland 20.3 18.5 12.3 Salivary gland 10.0 45.0 39.2 Leukaemia, HL-60 2.3 3.4 17.4 HeLa S3 3.5 20.2 14.8 Leukaemia, K-562 2.5 10.5 20.5 Leukaemia, MOLT-4 8.1 1.5 19.3 Burkitt, lympoma, Raji 6.5 8.0 27.5 Burkitt, lympoma, Daudi 1.1 1.4 25.6 Colorectal adenocacrinoma, SW480 5.1 9.4 8.1 Lung carcinoma, A549 0.7 1.8 4.3 Foetal brain 1.9 12.5 34.7 Foetal heart 3.9 22.5 8.4 Foetal kidney 9.7 54.1 5.6 Foetal liver 30.2 29.3 15.9 Foetal spleen 1.9 41.7 8.2 Foetal thymus 6.6 41.7 14.1 Foetal lung 8.5 24.1 16.0 Table 4. Kinetic parameters of wild-type and N-terminal deletion mutant. PNPO activities of wild-type and deletion mutant were measured in 0.1 M Tris/HCl at pH 8.4. Data shown are the average of three determinations ± SD. Enzyme Compound K m or K i a (l M ) V max (lmolÆmin )1 Æmg )1 ) k cat /K m ( M )1 ÆS )1 ) Wild-type PNP 2.1±0.2 0.10±0.06 5.2 · 10 4 PMP 6.2±0.3 0.05±0.01 8.2 · 10 3 PLP 3.8 D1–56 PNP 6.2±0.2 0.21±0.02 3.1 · 10 4 PMP 20.8±0.4 0.08±0.01 3.6 · 10 3 PLP 23.0 a Inhibition constant for product PLP determined with the sub- strate PNP. 2458 J. H. Kang et al. (Eur. J. Biochem. 271) Ó FEBS 2004 than that of the full-length PNPO. Thus, the value for the specificity constant (k cat /K m ) is compensated. PLP is a competitive inhibitor, and the K i values for the wild-type enzyme and D1–56 were 3.8 and 23 l M , respectively. Since the mechanism of PNPO is not yet fully understood, we cannot explain the changes in kinetic parameters. The N-terminal segment, however, would remain flexible and disordered in a solution, and it would form a lid over the active site [23]. This may play at least a partial role in binding and catalytic activity. Further truncation (D1–72) resulted in completely abol- ished enzymatic activity, indicating that the first highly conserved helix segment (residues 57–72) is required for activity. Previous studies showed that the peptide fragment of approximately two-thirds of the molecular mass yielded by a limited chymotryptic cleavage of sheep PNPO endowed with full catalytic activity [36]. This discrepancy may be due to a sequence difference between species or a disturbance in the folding process during expression caused by a missing structural unit. The presence of the first helical sequence might be solely structural, as it does not have a direct interaction with either PLP or FMN [23]. In addition, a deletion of 25 residues at the C terminus (D238–262) resulted in essentially inactive enzymes, indicating that this region is required for function. Conclusions In this report, we have described the genomic organization of PNPO, tissue distribution and deletion mutagenesis. (1) The human PNPO gene is composed of seven exons and six introns spanning  7.7 kb of the genomic DNA. The 5¢-flanking region has the characteristic features of housekeeping genes. Due to alternate usage of polyadeny- lation sites, two species of mRNA existed in all examined tissue. Nevertheless, no protein isoforms were detected. Fig. 5. Deletion analysis of recombinant PNPO. (A) Expression and purification of recombinant human PNPO. SDS/PAGE analysis (12% acrylamide)ofcrudecellextractsofE. coli BL21(DE3) containing the expression vector without and with the coding sequences for the wild-type or mutants. Lane M, Low molecular mass standards (Bio-Rad); lane 1, crude extracts from cultured cells harbouring pET28a; lane 2, cells containing pET28a/PNPO in the presence of 1 m M isopropyl thio-b- D -galactoside; lane 3, purified recombinant PNPO from Ni 2+ resin; lanes 4–6, purified deletion mutants: D1–56, D1–72 and D238–262, respectively. (B) Left, schematic structure of wild-type PNPO and the N- and C-terminal deletion mutants used in this study. Numbers refer to the amino acid position along the primary sequence of PNPO. Right, the effect of N- and C-terminal deletion on PNPO activity was expressed as a percentage of enzymatic activity in wild-type enzyme. Solid black and crosshatched bars are for substrate PNP and PMP, respectively. The results shown are the means ± SD from triplicate assays. Ó FEBS 2004 Human pyridoxine 5¢-phosphate oxidase (Eur. J. Biochem. 271) 2459 (2) The widespread distribution of PNP oxidase mRNA in human tissue agrees with its essential function in vitamin B 6 homeostasis. Three key enzymes for vitamin B 6 metabolism ) PNPO, PDXK and PDXP ) have remark- ably different expression profiles. (3) The catalytic core of PNPO was determined by sequential deletion mutants. 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