Báo cáo khoa học: Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5¢-phosphate oxidase pptx

Genomic organization, tissue distribution and deletion mutationof human pyridoxine 5¢-phosphate oxidase Jeong Han Kang1, Mi-Lim Hong1, Dae Won Kim2, Jinseu Park2, Tae-Cheon Kang3, Moo Ho

Genomic organization, tissue distribution and deletion mutation

of human pyridoxine 5¢-phosphate oxidase

Jeong Han Kang1, Mi-Lim Hong1, Dae Won Kim2, Jinseu Park2, Tae-Cheon Kang3, Moo Ho Won3,

Nam-In Baek4, Byung Jo Moon1, Soo Young Choi2and Oh-Shin Kwon1

1

Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu, Korea;2Department of Genetic Engineering, Division of Life Sciences, and3Department 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 B6 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 B6, 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 funcregula-tion [3].The enzymes that are

conventionally involved in vitamin B6metabolism 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¢-phos5¢-phos-phate (PMP) to PLP, with O2 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 colienzyme 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)

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 organizacharacteriza-tion, 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 transcripinvestiga-tion of human PNPO.In addiinvestiga-tion, the

minimum size necessary for enzymic function was

deter-mined by deletion mutagenesis

Materials and methods

Materials

A Marathon-ReadyTMcDNA library from human brain, a

multiple tissue Northern blot (MTNTMBlot) and a dot blot

array (MTETM 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-32P]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

NCBIBLASTsearches revealed an expressed sequence tag

(EST) clone (GenBankTM 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

94C for 1 min, annealing 55 C for 1 min and extension

72C for 2 min.The PCR product was cloned into the

pGEM-T vector (Promega) and sequenced (GenBankTM/ EBI accession number AF468030)

To facilitate expression vector construction, a BamHI recognition site was introduced at both ends of the ORF by PCR with primers shown in Table 1 The PCR mixture was 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 (GenBankTM/EBI accession number AF468030) was used to query human genome sequences usingBLASTN 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 silicoby using theMATINSPECTOR PROFESSIONALprogram

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 programCPGPLOTwas 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 65C for 1 h

in ExpressHybTM Hybridization solution (Clontech).The filters were then hybridized at 65C for 16 h with

32P-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

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.

Reverse 5¢-GGAAGCTTAGTTAAGGTGCAAGTCTCTC-3¢ HindIII

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.

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 37C.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 37C 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 H2O2] for

5 min at room temperature

Expression inE 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 37C in Luria–

Bertani medium with 50 lgÆmL)1kanamycin.When that

culture had grown to an A600 of 0.5, isopropyl

thio-b-D-galactoside was added to a final concentration of 1 mM

After inducing the expression of the PNPO protein for 3 h

at 37C, cells were harvested by centrifugation (10 000 g at

4C for 10 min), and the pellet was suspended in lysis

buffer (20 mM Tris/HCl pH 7.4, 1 mM EDTA, 200 mM

NaCl, 10 mM 2-mercaptoethanol, 0.5 mM

phenyl-methylsulfonyl fluoride).The cell suspension was sonicated,

and the lysate was cleared by centrifugation at 12 000 g and

4C for 20 min.The supernatant was then poured into the

column loaded with nickel-nitrilotriacetic acid agarose

(Qiagen), washed with Tris buffer containing 40 mM

imidazole, and protein was eluted with 200 mMimidazole

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 Tris/HCl pH 8.4 containing 0.1 m PNP.At this

wavelength, the Schiff base formed between Tris and PLP has an extinction coefficient of 5900M )1Æcm)1.One unit of specific activity is defined as the amount of protein that catalyses the formation of 1 lmol PLPÆmin)1at 25C.The value of Km and kcat 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, aBLASTanalysis (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.SCANPROSITEsoftware analysis byEXPASY 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 CGobs/CGexpratio in excess of 0 6 and a

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-INSPECTORsoftware (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¢- and 3¢-UTR sequences.The locations of CpG islands are indexed relative to the start codon, and indicated by the open boxes with numbers.

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

m30 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

PNPOgene 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

VIIa (1662) AGATTA

VIIb (2700) ATTGAT

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 with32P-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.

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

BLASTanalysis 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 (MTETM) 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 Lane M, Molecular mass standards; lane 1, brain; lane 2, liver; lane 3, lung; lane 4, prostate; lane 5, human breast cancer (MCF-7); lane 6, human uterine carcinoma (HL3T1); lane 7, stomach tissue.

mRNA expression levels in selected tissue for each enzyme

are shown in Table 3.Consistent with their ubiquitous role

in vitamin B6metabolism, 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.

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

Kmof 2.1 lM and 6.2 lMwere 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)

Vmaxvalues of 0.10 and 0.05 lmolÆmin)1Æmg)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

Kmvalue 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

Cerebral cortex 25.9 27.0 100.0

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

Cerebellum, left 13.3 21.9 90.5

Cerebellum, right 25.0 31.1 91.1

Corpus callosum 18.4 19.8 37.7

Caudate nucleus 19.0 28.4 74.2

Medulla oblongate 5.0 15.8 41.7

Accumbens nucleus 5.2 19.4 70.8

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

Colon, ascending 1.2 5.7 21.9

Colon, transverse 7.9 8.0 21.6

Colon, desending 2.8 9.4 6.5

Skeletal muscle 34.1 15.4 20.7

Peripheral blood leukocyte 0.4 26.3 13.3

Table 3 (Continued).

PNPO PDXK PDXP

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

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

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 ia

(l M )

V max

(lmolÆmin)1Æmg)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

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.

than that of the full-length PNPO.Thus, the value for the

specificity constant (kcat/Km) is compensated.PLP is a

competitive inhibitor, and the Ki values for the wild-type

enzyme and D1–56 were 3.8 and 23 lM, 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) of crude cell extracts of E 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.

(2) The widespread distribution of PNP oxidase mRNA

in human tissue agrees with its essential function in

vitamin B6homeostasis.Three key enzymes for vitamin B6

metabolism) PNPO, PDXK and PDXP ) have

remark-ably different expression profiles.(3) The catalytic core

of PNPO was determined by sequential deletion mutants

The deletion of the N-terminal 56 residues did not affect

binding of coenzyme, or catalytic activity, whereas deletion

of the C-terminal region resulted in an inactive enzyme

The results obtained here will contribute directly to future

studies aimed at a better understanding of the catalytic

mechanism of PNPO and vitamin B6 metabolism.In

particular, the tissue-specific effects on mRNA stability

and the regulatory mechanism governing the PNPO gene

expression require further investigation

Acknowledgements

This work was supported by Grant R01-2002-000-00008-0 from Basic

Research Program of the Korea Science & 21st Century Brain Frontier

Research Grant (M103KV010019–03K2201-01910) from the Ministry

of Science and Technology, Korea.

References

1.Snell, E E.(1990) Vitamin B6 and decarboxylation of histidine.

Ann NY Acad Sci 585, 1–12.

2 McCormick, D B.& Merrill, A H.(1980) Pyridoxamine

(pyri-doxine) 5¢-phosphate oxidase.In Vitamin B 6 Metabolism and Role

in Growth (Tryfiates, G.P., ed.), pp 1–26 Food and Nutrition

Press, Westport, CT.

3 Robson, L.C & Schwartz, M.R (1975) Vitamine B 6 deficiency

and the lymphoid system I.Effects on cellular immunity and in

vitro incorporation of 3 H-uridine by small lymphocytes Cell.

Immunol 16, 135–144.

4 McCormick, D.B., Gregory, M.E & Snell, E.E (1961) Pyridoxal

phosphokinase I: assay, distribution, purification and properties.

J Biol Chem 236, 2076–2084.

5 Hanna, M.C., Turner, A.J & Kirkness, E.F (1997) Human

pyridoxal kinase.cDNA cloning, expression, and modulation by

ligands of the benzodiazepine receptor J Biol Chem 272, 10756–

10760.

6 Kwok, F.& Churchich, J.E.(1992) Pyrdoxine-5¢-P oxidase.In

Chemistry and Biochemistry of Flavoenzymes (Muller, F., ed.),

Vol.3, pp.1–20.CRC Press, London.

7 Ngo, E O , LePage, G R , Thanassi, J W , Meisler, N & Netter,

L.M (1998) Absence of PNP oxidase (PNPO) activity in

neo-plastic cells: isolation, characterization, and expression of PNPO

cDNA Biochemistry 37, 7741–7748.

8.Fonda, M L.(1992) Purification and characterization of vitamine

B 6 -phospate phosphatase from human erythrocytes J Biol.

Chem 267, 15978–15983.

9 Jang, Y M , Kim, D W , Kang, T C , Won, M H , Baek, N I ,

Moon, B.J., Choi, S.Y & Kwon, O.S (2003) Human Pyridoxal

Phosphatase: Molecular cloning, functional expression and tissue

distribution J Biol Chem 278, 50040–50046.

10 Choi, J.D., Bowers-Komro, D.M., Davis, M.D., Edmondson,

D.E & McCormick, D.B (1983) Kinetic properties of

pyridox-amie (pyridoxine) 5¢-phosphate oxidase from rabbit liver J Biol.

Chem 258, 840–845.

11.Wada, H.& Snell, E E.(1961) The enzymatic oxidation of

pyri-doxine and pyridoxamine-phosphates J Biol Chem 236, 2089–

2095.

12 Di Salvo, M , Yang, E , Zhao, G , Winkler, M E & Schirch, V.

(1998) Expression, purification and characterization of

recom-binant Escherichia coli pyridoxine 5¢-phosphate oxidase Protein Express Purif 13, 349–356.

13 Kazarinoff, M.N & McCormick, D.B (1975) Rabbit liver pyri-doxamine (pyridoxine) 5¢-phosphate oxidase J Biol Chem 250, 3436–3442.

14.Churchich, J E.(1984) Brain pyridoxine-5-phosphate oxidase: a dimeric enzyme containing one FMN site Eur J Biochem 138, 327–332.

15 Choi, S.Y., Churchich, J.E., Zaiden, E & Kwok, F (1987) Brain pyridoxine-5¢-phosphate oxidase: modulation of its catalytic activity by reaction with pyridoxal 5¢-phosphate and analogs.

J Biol Chem 262, 12013–12017.

16 Tsuge, H., Itoh, K., Akatsuka, F., Okada, T & Ohashi, K (1987) Inactivation of pyridoxamine-5¢-P oxidase by aliphatic primary amines Biochem Int 6, 743–749.

17 Notheis, C., Drewke, C.& Leistner, E.(1995) Purification and characterization of the pyridoxol-5¢-phosphate: oxygen oxido-reductase (deaminating) from Escherichia coli Biochim Biophys Acta 1247, 265–271.

18.Zhao, G.& Winkler, M.(1995) Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5¢-phosphate oxidase of Escherichia coli K-12 J Bacterol 177, 883–891.

19 Di Salvo, M.L., Safo, M.K., Musayev, F.N., Bossa, F & Schirch, V (2003) Structure and mechanism of Escherichia coli pyridoxine 5¢-phosphate oxidase Biochim Biophys Acta 1647, 76–82.

20 Safo, M K , Mathews, I , Musayev, F N , Di Salvo, M L , Thiel, D.J., Abraham, D.J & Schirch, V (2000) X-ray structure of Escherichia coli pyridoxine 5¢-phosphate oxidase complexed with FMN at 1.8 A˚ resolution Structure 8, 751–762.

21 Safo, M.K., Musayev, F.N., De Salvo, M.L & Schirch, V (2001) X-ray structure of Escherichia coli pyridoxine 5¢-phosphate oxi-dase complexed with pyridoxal 5¢-phosphate at 2.0A˚ resolution.

J Mol Biol 310, 817–826.

22 Di Salvo, M.L., Ko, T.P., Musayev, F.N., Raboni, S., Schirch, V.

& Safo, M.K (2002) Active site structure and stereospecificity of Escherichia coli pyridoxine-5¢-phosphate oxidase J Mol Biol.

315, 385–397.

23 Musayev, F.N., Di Salvo, M.L., Ko, T.P., Schirch, V & Safo, M.K (2003) Structure and properties of recombinant human pyridoxine 5¢-phosphate oxidase Protein Sci 12, 1455–1463.

24 Quandt, K., Frech, K., Karas, H., Wingender, E & Werner, T (1995) MatInd and MatInspector: new fast and versatile tools from detection of consensus matches in nucleotide sequence data Nucleic Acids Res 23, 4878–4884.

25.Gardiner-Garden, M.& Frommer, M.(1987) CpG islands in vertebrate genomes J Mol Biol 196, 261–282.

26.Rice, P , Longden, I.& Bleasby, A.(2000) EMBOSS: the Euro-pean Molecular Biology Open Software Suite Trends Genet 16, 276–277.

27 Lee, H.-S., Moon, B.J., Choi, S.Y & Kwon, O.S (2000) Human pyridoxal kinase: Overexpression and properties of the recom-binant enzyme Mol Cells 10, 452–459.

28 Bahn, J.H., Kwon, O.S., Joo, H.M., Jang, S.H., Park, J., Hwang,

I K , Kang, T C , Won, M H , Kwon, H Y , Kwok, F , Kim, H B , Cho, S.W & Choi, S.Y (2002) Immunohistochemical studies of brain pyridoxine-5¢-phosphate oxidase Brain Res 925, 159–168.

29 Weis, L.& Lindenberg, D.(1992) Transcription by RNA poly-merase II: initiator-directed formation of transcription-competent complexes FASEB J 6, 3300–3309.

30.Wickens, M.(1990) How the messenger got its tail: addition of poly (A) in the nucleus Trends Biochem Sci 15, 277–281.

31 Grzybowska, E.A., Wilczynska, A & Siedlecki, J.A (2001) Reg-ulatory functions of 3¢ UTRs Biochem Biophys Res Commun.

288, 291–295.

32 Qu, X., Qi, Y.& Qi, B.(2002) Generation of multiple mRNA transcripts from the novel human apoptosis-inducing gene hap

by alternative polyadenylation utilization and the translational

activation function of 3¢ untranslated region Arch Biochem.

Biophys 400, 233–244.

33 Snell, E.E & Haskell, B.E (1971) The metabolism of vitamine B 6

In Metabolism of Vitamins and Trace Elements (Florkin, M &

Stotz, E.H., eds), Vol.21, pp.47–71.Elsevier Scientific Publishing

Co, Amsterdam.

34 Anderson, B.B., Newmark, P.A & Rawlins, M (1974) Plasma

binding of vitamine B6 compound Nature 250, 502–504.

35 Lumeng, L.& Li, T.K.(1975) Characterization of the pyridoxal 5¢-phosphate and pyridoxamine 5¢-phosphate hydrolase activity in rat liver.Identity with alkaline phosphatase.J Biol Chem 250, 8126–8131.

36 Kwon, O.S., Kwok, F & Churchich, J.E (1991) Catalytic and regulatory properties of native and chymotrypsin-treated pyridoxine-5-phosphate oxidase J Biol Chem 266, 22136– 22140.