Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M309619200 on September 30, 2003

J. Biol. Chem., Vol. 278, Issue 50, 50040-50046, December 12, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/50/50040    most recent
M309619200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jang, Y. M.
Right arrow Articles by Kwon, O.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jang, Y. M.
Right arrow Articles by Kwon, O.-S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Human Pyridoxal Phosphatase

MOLECULAR CLONING, FUNCTIONAL EXPRESSION, AND TISSUE DISTRIBUTION*

Young Min Jang{ddagger}, Dae Won Kim§, Tae-Cheon Kang¶, Moo Ho Won¶, Nam-In Baek||, Byung Jo Moon{ddagger}, Soo Young Choi§**, and Oh-Shin Kwon{ddagger}{ddagger}{ddagger}

From the {ddagger}Department of Biochemistry, Kyungpook National University, Taegu 702-701, Korea, the §Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea, the Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, Korea, and the ||Graduate School of Biotechnology & Plant Metabolism Research Center, Kyunghee University, Suwon 449-701, Korea

Received for publication, August 29, 2003 , and in revised form, September 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyridoxal phosphatase catalyzes the dephosphorylation of pyridoxal 5'-phosphate (PLP) and pyridoxine 5'-phosphate. A human brain cDNA clone was identified to the PLP phosphatase on the basis of peptide sequences obtained previously. The cDNA predicts a 296-amino acid protein with a calculated Mr of 31698. The open reading frame is encoded by two exons located on human chromosome 22q12.3, and the exon-intron junction contains the GT/AG consensus splice site. In addition, a full-length mouse PLP phosphatase cDNA of 1978 bp was also isolated. Mouse enzyme encodes a protein of 292 amino acids with Mr of 31512, and it is localized on chromosome 15.E1. Human and mouse PLP phosphatase share 93% identity in protein sequence. A BLAST search revealed the existence of putative proteins in organism ranging from bacteria to mammals. Catalytically active human PLP phosphatase was expressed in Escherichia coli, and characteristics of the recombinant enzyme were similar to those of erythrocyte enzyme. The recombinant enzyme displayed Km and kcat values for pyridoxal of 2.5 µM and 1.52 s–1, respectively. Human PLP phosphatase mRNA is differentially expressed in a tissue-specific manner. A single mRNA transcript of 2.1 kb was detected in all human tissues examined and was highly abundant in the brain. Obtaining the molecular properties for the human PLP phosphatase may provide new direction for investigating metabolic pathway involving vitamin B6.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyridoxal 5'-phosphate (PLP)1 is the coenzymatically active form of vitamin B6 and plays an important role in maintaining the biochemical homeostasis of the body (1, 2). Thus, the other nutritionally available vitamer forms in this family must be converted to PLP. The enzymes that are conventionally involved in the metabolism are an ATP dependent pyridoxal kinase (EC 2.7.1.35 [EC] ) (3, 4) and a flavin mononucleotide-dependent pyridoxine 5'-phosphate (PNP) oxidase (EC 1.4.3.5 [EC] ) (5, 6). On the other hand, the preferred degradation route from PLP to 4-pyridoxic acid involves the dephosphorylation of PLP by the phosphatase (7) followed separately by the actions of aldehyde oxidase and {beta}-nicotinamide adenosine dinucleotide-dependent dehydrogenase (8, 9).

The major pathways of vitamin B6 metabolism have been established for decades, but little is known of how the concentration of PLP is controlled in mammalian tissues. Studies on the homeostasis have shown that there is not a single mechanism. The factors for the regulation of PLP may include catabolism of PLP by PLP phosphatase, activities of pyridoxal kinase and PNP oxidase, degree of protein binding of the synthesized coenzyme, and transport of the precursors (10, 11). Although PNP oxidase plays a kinetic role in regulating the level of PLP formation (12, 13), PLP availability is mainly dependent on protein binding and phosphatase action (10, 14, 15). Thus, the phosphatase action may be important in the regulation of PLP concentration.

A PLP-specific phosphatase has been purified from human erythrocytes (16). The molecular properties including the molecular weight, pH optimum, substrate specificity, and metal requirement indicate that the PLP phosphatase distinct from other known phosphatases. Alkaline phosphatase (EC 3.1.3.1 [EC] ) and acid phosphatase (EC 3.1.3.2 [EC] ) also hydrolyze PLP and pyridoxamine 5'-phosphate (PMP), but they have broad substrate specificity for phosphomonoesters (17, 18). Alkaline phosphatase is resistant to fluoride and inhibited by L(+)-tartrate and levamisole, whereas the PLP-specific phosphatase not. Acid phosphatase is not inhibited by fluoride and EDTA. The PLP phosphatase is most active at physiological pH and requires Mg2+ for activity (19). It is a dimer with a molecular mass of 64 kDa, and has a Km value of ~1 µM for PLP, which is 10-fold lower than that reported with other phosphatases (16). Data obtained in chemical modification studies revealed that PLP phosphatase has essential residues such as cysteine, arginine, histidine, and a carboxylate group at or near the active site (20–22).

Although the PLP-specific phosphatase has been purified and characterized, a cDNA encoding PLP phosphatase has not yet been identified. Here we cloned cDNAs encoding PLP phosphatase from human and mouse. In addition, we describe the high efficiency expression of the protein in Escherichia coli with its kinetic properties and the tissue-specific expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—We purchased a human brain cDNA library as well as a dot blot array containing poly(A)+ RNAs from human tissues from Clontech (Palo Alto, CA). Restriction endonuclease and other cloning reagents were purchased from New England Biolabs, Inc. (Beverly, MA) or Promega. Double-stranded DNA probes were radiolabeled with [{alpha}-32P]dCTP (3000 Ci/mmol) from Amersham Biosciences using a commercial random priming kit (Amersham Biosciences). All other reagentgrade chemicals were obtained from standard suppliers.

Identification and Cloning of Human Brain PLP Phosphatase (hPLPP) cDNA—BLAST searches, conducted with PLP phosphatase peptides (AQGVLFDCDGVL and AVLVGYDEHFSFAK) as query sequences, revealed a full match with the predicted amino acid sequence of a cDNA (GenBankTM/EBI accession number NM020315). This clone was used to design PCR primers for the cloning of human PLP phosphatase cDNA.

We employed 5'-rapid amplification of cDNA ends (RACE) using hPLPP-specific primer 1 (5'-CGATGCTGAAGTTCTCCGTGATGCAC-3'), AP1 anchor primer, and Marathon Ready cDNA (human whole brain, Clontech) as a template. PCR was carried out with betaine in a GeneAmp PCR system 2400 (PerkinElmer Life Sciences) for 30 cycles of denaturation (94 °C, 30 s annealing; 60 °C, 1 min; 72 °C, 2 min extension). The resulting PCR product of 737 bp fragment revealed that the 5'-untranslated region is completely matched except 9 bp shorter than the cDNA from lung carcinoma. The full-length open reading frame (ORF) of 891 bp was amplified using the hPLPP-specific primers (sense, 5'-GGATCCGGCTGCATGGCGCGCTGCGA-3'; antisense, 5'-GGATCCGGGGTGG GCCTGTGGCTGCAG-3'). The PCR product was cloned into the pGEM-T vector (Promega) and sequenced (GenBankTM accession number AY125047 [GenBank] ).

A multiple protein sequence alignment of the human PLP phosphatase clone, along with the sequences most closely related to it, was performed using the ClustalW (version 1.74) program (23), and the resulting aligned sequence were shaded using Boxshade. Identification of the chromosomal localization of the human PLP phosphatase was performed by a BLASTN search of the human genome sequences of the National Center for Biotechnology Information (NCBI).

Cloning of Mouse Brain PLP Phosphatase (mPLPP) cDNA—We also identified a mouse expressed sequence tag (EST) clone (GenBankTM accession number AK043228 [GenBank] ) with high similarity to human PLP phosphatase. To obtain a full-length cDNA corresponding to this EST clone, 5'-RACE and 3'-RACE were performed using mouse brain cDNA (Seegene, Seoul, Korea) according to the manufacturer's protocol. Two consecutive PCR reactions using Ex Taq polymerase (Takara) were performed as follows: (1) 5'-RACE, 94 °C for 3 min followed by 30 cycles of 94 °C for 1 min and 63 °C for 40 s, followed by primer extension at 72 °C for 5 min with 5'-RACE primer (5'-GTCTACCAGGCATTCGCTTCAT-3') and mPLPP-specific primer 1 (5'-ATCCCAGCCTCCCTTCACT-3'); and (2) 3'-RACE, 94 °C for 3 min followed by 30 cycles of 94 °C for 1 min, 56 °C for 40 s, and primer extension at 72 °C for 5 min with 3'-RACE primer (5'-CTGTGAATGCTGCGACTACGAT-3') and mPLPP-specific primer 2 (5'-ATGGAAGGGAGGCTGGGATCCAGG-3'). The PCR products were cloned and sequenced.

Expression in E. coli and Purification of Human PLP Phosphatase— The PLP phosphatase cDNA was cloned between the BamHI of the bacterial expression vector pQE30 (Qiagen) after PCR amplification. Transformants of E. coli M15/pRER4 with the resulting pQE30-hPLPP construct were grown at 37 °C in 200 ml of LB medium with 100 µg/ml ampicillin and 25 µg/ml kanamycin. The plasmid pREP4 constitutively expresses the Lac repressor protein encoded by the lacI gene to reduce the basal level of expression (Qiagen). When that culture had grown to an A600 of 0.6, isopropyl {beta}-D-thiogalactopyranoside was added to a final concentration of 1 mM. After inducing the expression of the PLP phosphatase protein for 16 h at 25 °C cells were harvested, washed, and resuspended in 20 ml of 20 mM Tris-HCl buffer, pH 7.4, containing 200 mM NaCl and 20 mM imidazole.

The cell suspension was sonicated, and the lysate was cleared by centrifugation at 10,000 x g and 4 °C for 30 min. The supernatant was then poured into the column loaded with the nickel-nitrilotriacetic acid agarose (Qiagen), washed with Tris buffer containing 40 mM imidazole, and eluted protein was eluted with 200 mM imidazole. The purity of the eluted protein was evaluated by 12% SDS-PAGE using Coomassie blue staining to visualize the protein.

Enzyme Assay and Kinetic Characterization—The enzymatic activity of PLP phosphatase was measured at pH 7.4 in 40 mM triethanolamine-HCl. The rate of production of pyridoxal from PLP was measured by following the decrease in absorbance at 390 nm for at least 3 min. The initial velocity data were fitted by a least-squares method to the Line-weaver-Burk transformation of the following equation,

where [S] represents the concentration of the varied substrate PLP, and Km represents the Michaelis constant.

A molecular weight of 64,000 for PLP phosphatase with a specific activity of 1.4 µmol·min–1mg–1 was used in the calculations of molar enzyme concentrations. One unit of specific activity is defined as the amount of protein that catalyzes the formation of pyridoxal/min from PLP. PLP is known to have an extinction coefficient of 4900 cm–1 M–1 at pH 7. Protein concentrations were determined by the Bradford assay using the reagent and micro procedure from Pierce Chemical Co. Bovine serum albumin was used as a standard.

In addition, hydrolysis of all other substrates was measured in 40 mm triethanolamine-HCl, pH 7.4, containing 4 mM MgCl2. The release of free phosphate from potential substrates was measured colorimetrically as the molybdate complex with malachite green (16, 24).

Multiple Tissue Expression Array—A Northern filter containing 8 human tissue-specific poly(A)+ RNAs and a dot blot array containing human poly(A)+ RNAs from various adult tissues, fetal tissues, and cancer cell lines were prehybridized at 65 °C for 1 h in ExpressHybTM hybridization solution (Clontech). The filters were then hybridized at 65 °C for 16 h with 32P-labeled cDNA probe containing the complete ORF. 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. For scanning densitometry, the blot was scanned and BioLab Image software was used to quantify the signals. Blots were reprobed with {beta}-actin as a loading control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Cloning of the PLP Phosphatase cDNA from Human and Mouse—Using two tryptic peptide sequences from the erythrocytes PLP phosphatase previously reported by Gao and Fonda (21), we conducted NCBI BLAST searches of the human EST data base. A cDNA clone encoding both peptide sequences, designated a hypothetical protein dj37E 16.5 from lung carcinoma (GenBankTM/EBI accession number NM020315) was identified. When the tryptic peptide sequences were aligned with the deduced amino acid sequence there was a perfect match, confirming that the cDNA in the EST GenBankTM data base codes for PLP phosphatase.

We used the sequence information for designing specific primers for PCR amplification utilizing a human brain cDNA library. Sequencing of this clone revealed that the brain cDNA is identical to the hypothetical protein from lung carcinoma. The ORF encodes a 296-amino acid protein with a molecular mass of 31698 Da. A computer calculation reveals that the isoelectric point for the protein is 6.12. ScanProsite software analysis by ExPASy showed that the deduced human protein has the following putative post-translational modification sites: 2 N-glycosylation sites, 7 phosphorylation sites, and 4 N-myristoylation sites.

In addition, we cloned a complete mouse homologue by 5'-RACE and 3'-RACE-PCR using an EST sequence (GenBankTM/EBI accession number AK043228 [GenBank] ). As shown in Fig. 1, the longest cDNA contains 1978 bp consisting of a 879-bp ORF, a 13-bp 5'-untranslated region, and a 1086-bp 3'-noncoding region. The 3'-end of the sequence contains a poly(A) stretch, preceded by a putative polyadenylation signal AATAAA. The open reading frame encodes a 292-amino acid protein with a predicted molecular mass of 31512 Da and pI of 5.53. The deduced protein has a putative N-glycosylation site, 5 phosphorylation sites, and 4 amidation sites.



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 1.
Nucleotide and deduced amino acid sequence of a full-length cDNA encoding mouse PLP phosphatase (GenBankTM accession number AY366300 [GenBank] ). The predicted amino acid sequence of PLP phosphatase is shown below the nucleotide sequence. The termination codon is marked by an asterisk, and the polyadenylation signal is double underlined. Putative glycosylation sites (g), phosphorylation sites (p), and N-myristoylation sites (m) in mouse PLP phosphatase are indicated. A potential exon/intron junction is also indicated by a slash. The numbers on the right refer to nucleotide and amino acid positions.

 
Using BLAST searches of the EST data bases, we found putative PLP phosphatases in various species. Multiple alignment of the protein sequence of PLP phosphatase with other homologous proteins or putative proteins was performed using the ClustalW program as shown in Fig. 2. All of the PLP phosphatases are highly homologous at the amino acid sequence level. The mouse PLP phosphatase sequence shows a high degree of similarity (94% identity and 98% similarity) with the human enzyme. The sequence identities of human PLP phosphatase to the hypothetical proteins from Danio rerio, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and E. Coli are 36, 28, 26, 25, 31, and 27%, respectively, and similarities share 77, 65, 68, 60, 70, and 66%, respectively. This result suggests that PLP phosphatase is highly homologous and widely expressed throughout evolution. The tryptic sequences from human erythrocytes enzyme that are perfectly matched with the deduced amino sequence are indicated by asterisks in Fig. 2.



View larger version (102K):
[in this window]
[in a new window]
  		FIG. 2.
Sequence alignment of human and mouse PLP phosphatase with homologous proteins. Full-length Homo sapiens hPLPP and Mus musculus mPLPP sequences were aligned with those of homologous proteins or putative proteins using the Clustal W program (version 1.74). EST sequences (GenBankTM accession number in brackets) are from D. rerio (AAH45860 [GenBank] ), D. melanogaster (AAF49296 [GenBank] ), C. elegans (P19881 [GenBank] ), A. thaliana (BAA98057 [GenBank] ), S. cerevisiae (NP504509), and E. Coli (AAC09327 [GenBank] ). The introduced gaps are shown as hyphens, and aligned amino acids are boxed (black for identical residues and dark gray for similar residues). The tryptic fragment sequences determined from human erythrocytes enzyme are indicated by asterisks.

 
Genomic Organization and Chromosomal Localization—Using PLPP cDNA as a query sequence, a BLAST analysis of the genomic sequence available through NCBI Web site mapped the PLP phosphatase gene to human chromosome 22q12.3. Mouse PLP phosphatase gene is located on chromosome 15.E1 with a very similar genomic organization to that observed in human. The ORF of PLP phosphatase is encoded by two exons as shown in Fig. 3. The intron/exon junctions followed the GT/AG rule (25) and were flanked by conserved sequences. The lariate branch-point in the consensus sequence is located at 23 nucleotides upstream of the 3' splice site. The possible exon/intron junction site in mouse cDNA is also indicated by a slash in Fig. 1.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 3.
Genomic organization of human and mouse PLP phosphatase genes. Schematic representation of human (A) and mouse PLP phosphatase genes (B) arranged as two exons. Exon regions are denoted by boxes. Shadowed boxes represent ORF, and open boxes denote the 5'- and 3'-untranslated regions. Black lines indicate the introns. The consensus intron/exon junction sequences are underlined, and the intron/exon borders are marked by slashes.

 
Functional Expression and Catalytic Properties of the Human Recombinant Enzyme—After E. coli M15/pQE30-hPLPP was induced with isopropyl {beta}-D-thiogalactopyranoside, the His6-tagged recombinant PLP phosphatase yielded a major band with an apparent molecular mass of 32 kDa on an SDS-PAGE gel corresponding to about 15% of the total applied protein (Fig. 4). Most of the expressed protein was found in the soluble extract after lysis of the cells. As shown in Table I, the crude extraction from 200 ml of cells contained ~14.5 mg of total protein and 0.14 units/mg of PLP phosphatase activity. Purification by binding to Ni2+-nitrilotriacetic acid-agarose resin and elution with buffer containing 200 mM imidazole resulted in a near homogeneous preparation. The purified recombinant protein had a specific activity of 1.4 units/mg and appeared as a single band with a molecular mass of 32 kDa agreeing with the value calculated from the deduced sequence (Fig. 4, lane 6).



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 4.
Expression and purification of recombinant human PLP phosphatase. Twelve percent of SDS-PAGE analysis of crude cell extracts of E. coli M15 containing the expression vector pQE30 without and with the coding sequence. Lane 1, low molecular mass standards (Bio-Rad Laboratory); lanes 2 and 3, crude extracts from cultured cells harboring pQE30 uninduced and induced with 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside, respectively; lanes 4 and 5, cells containing pQE30-hPLPP in the absence and presence of isopropyl-1-thio-{beta}-D-galactopyranoside, respectively; lane 6, purified recombinant PLP phosphatase from Ni resin (7 µg).

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Purification of human recombinant PLP-phosphatase

 
Activity of the human PLP phosphatase toward PLP, PNP, and PMP was determined (Table II). Recombinant human enzyme followed Michaelis-Menten kinetics with respect to PLP, and the Michaelis constant and kcat value for this reactant was 2.5 µM and 1.52 s–1, respectively. Km for PNP and PMP were 43 and 81 µM, respectively, and kcat values were 1.25 and 0.45 s–1, respectively, whereas activity with p-nitrophenyl phosphate was very low. Thus, the specificity constant of the recombinant PLP phosphatase was highest with PLP followed by PNP. The recombinant enzyme requires a divalent ion for activity, and Mg+2 is the most effective in catalyzing the dephosphorylation of PLP.


View this table:
[in this window]
[in a new window]
  		TABLE II
Kinetics of purified PLP-phosphatase with various substrates

Reaction mixtures containing 4 mM MgCl2, four different concentrations of indicated compounds, and 20 {approx} 300 µM purified PLPP in 40 mM TEA-HCI (pH 7.4), were incubated at 37°C for 3 min. Km and Vmax values, fitted with linear regression analysis, are means ± S.E.

 
To investigate the state of aggregation of recombinant human PLP phosphatase, the purified enzyme was examined by FPLC gel filtration at pH 7.4 in 50 mM triethanolamine-HCl buffer. The elution profile of the enzyme exhibits a symmetrical peak characterized by a molecular mass of about 60 kDa when the concentration of the recombinant protein in the fraction is around 10 µM (data not shown). These data clearly indicate that the recombinant enzyme is a dimer, agreeing with previous data for the erythrocyte enzyme.

Tissue-specific Expression of Human PLP Phosphatase—Dot blot array and Northern blot analyses were used to determine the expression pattern for PLP phosphatase in human tissues. As evident by multiple tissue expression (MTETM) array analysis, PLP phosphatase transcripts were detected in most tissues, indicating ubiquitous expression. However, the level of expression was markedly variable. PLP phosphatase was most highly represented in all the regions of central nerve system except the spinal cord (Fig. 5). The other major sites of expression were found in liver and testis. In fetus, expression levels of PLP phosphatase transcript showed a rather even distribution in all organs except brain. Like adult, fetal brain expressed PLP phosphatase transcript with a very high level. To determine the size of mRNA transcripts, Northern blots analyses were performed. A single 2.1-kb transcript was detected with variable intensity in all adult tissues examined: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (data not shown).



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 5.
Tissue-specific expression of the human PLP phosphatase mRNA. The human multiple tissue expression (MTETM) array was hybridized with a 32P-labeled human PLP phosphatase-specific cDNA probe. Tissue sources for the RNA are indicated below the blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we describe the first identification and characterization of cDNAs encoding a PLP phosphatase from human and mouse. A human cDNA clone of PLP phosphatase was identified by NCBI BLAST search on the basis of the amino acid sequences of two peptides from the purified human erythrocytes enzyme. Several lines of evidence confirm that the cDNA is indeed encoding the PLP phosphatase. First, the amino acid sequences of the two peptides of the purified protein are contained in the amino acid sequence deduced from the ORF (Fig. 2). Second, the molecular mass of the protein of 32 kDa calculated from the deduced cDNA sequence is identical to the values from SDS-polyacrylamide gel electrophoresis for the purified erythrocyte protein and the recombinant protein. Third, the kinetic parameters of the recombinant protein are similar to those of purified enzyme. The Km values for the substrate PLP (2.5 µM) are practically indistinguishable from those of the erythrocyte (1.5 µM) (16).

Human PLP phosphatase cDNA encodes a 296-amino acid protein with a predicted molecular mass of 31698 Da. The hydropathy profile of PLP phosphatase indicated a rather even distribution of hydrophobic and hydrophilic regions throughout the molecule (data not shown). Therefore, no evidence for integral membrane domains in PLP phosphatase was found. The isolated cDNA contains a short 5'-untranslated segment similar to lung carcinoma cDNA. No larger product has been generated in repeated RACE experiments using different primer sets. Furthermore, the 2.1-kb message determined by Northern analysis agrees well with the size of the full-length cDNA, suggesting that the 5'-end of the cDNA is at or near the transcription start site. Therefore, the cloned human brain cDNA may be complete. By contrast, analysis the EST clone for mouse PLP phosphatase shows that it was incomplete. The mouse EST sequence (GenBankTM/EBI accession number AK043228 [GenBank] ) contains several unidentified nucleotides and a stop codon in ORF, but no polyadenylate tail. Thus, we cloned a full-length cDNA of mouse PLP phosphatase by 5'- and 3'-RACE-PCRs, and the sequence has been deposited in GenBankTM/EBI as accession number AY366300 [GenBank] .

Comparison of the identified cDNA sequences with the genome sequences revealed the genomic structure and chromosomal localization. The PLP phosphatase gene is divided into two exons, and the splice site sequences are remarkably well conserved as shown in Fig. 3. In mammals two ends of the intron are conserved by a donor-site consensus sequence AG/GURAGU and an acceptor-site consensus sequence (Y)nNCAG/G, where R, Y, and N indicate purine, pyrimidine, and any nucleotide, respectively (25, 26). A branch-point consensus sequence, YNCUGAC, is usually about 15–40 nucleotides upstream of the 3' splice site, and the adenosine in the sequence forms the 2'–5' linkage in the lariat structure. In addition, the genomic sequences were examined for the presence of CpG islands using the CpG plot program in European Bioinformatics Institute (EBI), as defined by Gardiner-Garden and Frommer (27). The hPLPP gene contains a CpG island with a CGobs/CGexp ratio in excess of 0.8 and a GC content of 74% spanning a region from 343-bp upstream to 614-bp down-stream of the start codon, whereas the mPLPP gene has a CpG island extending from –282 bp to +643 bp with a CG content of 71%.

We also reported here the functional expression of human PLP phosphatase. The expressed protein was purified by a one-step affinity chromatographic method, and consequently endogenous PLP phosphatase produced by E. coli was eliminated. Enzymological analysis of the recombinant human protein expressed from this cDNA clone showed a high specificity for the hydrolysis of PLP. Characteristics of the recombinant enzyme were similar to those from the native enzyme purified from human erythrocytes, except kinetic values for PNP (16). Noteworthy is the relatively high Vmax and Km with PNP as a substrate. As shown in Table II, the Km values of recombinant enzyme for PNP and PLP were 43 and 2.5 µM, respectively, whereas the Vmax values for PNP were surprisingly high as 1.2 units/mg, which is almost same to the value for PLP (1.4 units/mg). Judging from the kinetic results, the catalytic efficiency (kcat/Km) for PNP was 20-fold lower than that of PLP. On the other hand, the kinetic values for the native enzyme were somewhat different (16). The enzyme had relatively low Km and Vmax values with PNP. Km values with respect to PNP and PLP were 5.2 and 1.5 µM, respectively, whereas the Vmax were 3.2 units/mg and 0.7 units/mg, respectively. In consequence, catalytic efficiency value for PNP was 17-fold low than that of PLP, which is practically indistinguishable from that of the recombinant enzyme.

An interesting aspect of our work is the finding that PLP phosphatase is differentially expressed in a tissue-specific manner. Based on the levels of mRNA expression, the wide-spread distribution of PLP phosphatase in human tissues is consistent with its essential role in cellular metabolism. In addition to its housekeeping role in PLP metabolism, however, PLP phosphatase may play a more specific role especially in brain, where its level is substantially higher. In this connection, it is should be noted that pyridoxal kinase is expressed in essentially all mammalian organs (4), whereas PNP oxidase is selectively present with the highest activity found in liver and kidney (28).

In conclusion, the results obtained in this study will provide valuable information on the action mechanism of human PLP phosphatase and the physiological role of this enzyme. The availability of the cDNAs encoding PLP phosphatase, and the recombinant enzyme will be helpful to expand our view of metabolic pathways that could involve PLP phosphatase and to evaluate its functional role in human cellular homeostasis.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY125047 [GenBank] and AY366300 [GenBank] .

* This work was supported by Grant R01-2002-000-00008-0 from the Basic Research Program of the Korea Science & Engineering Foundation and Korea Health 21 R&D Project Grant (02-PJ1-PG10-20706-0002) from the Ministry of Health and Welfare, Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence may be addressed. E-mail: sychoi{at}hallym.ac.kr. {ddagger}{ddagger} To whom correspondence may be addressed: Dept. of Biochemistry, Kyungpook National University, Taegu, 702-701, Korea. Tel.: 82-53-950-6356; Fax: 82-53-943-2762; E-mail: oskwon{at}knu.ac.kr.

1 The abbreviations used are: PLP, pyridoxal 5'-phosphate; PNP, pyridoxine 5'-phosphate; PMP, pyridoxamine 5'-phosphate; PLPP, PLP phosphatase; h, human; m, mouse; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Snell, E. E. (1990) Ann. N. Y. Acad. Sci. 585, 1–12[Medline] [Order article via Infotrieve]
  2. Meister, A. (1990) Ann. N. Y. Acad. Sci. 585, 13–31[Medline] [Order article via Infotrieve]
  3. McCormick, D. B., Gregory, M. E., and Snell, E. E. (1961) J. Biol. Chem. 236, 2076–2084[Free Full Text]
  4. Hanna, M. C., Turner, A. J., and Kirkness, E. F. (1997) J. Biol. Chem. 272, 10756–10760[Abstract/Free Full Text]
  5. Kwok, F., and Churchich, J. E. (1992) in Chemistry and Biochemistry of Flavoenzymes (Muller, F., ed) Vol. 3, pp. 1–20, CRC Press, London
  6. Ngo, E. O., LePage, G. R., Thanassi, J. W., Meisler, N., and Netter, L. M. (1998) Biochemistry, 37, 7741–7748[CrossRef][Medline] [Order article via Infotrieve]
  7. Lumeng, L., and Li, T. K. (1980) in Vitamin B6 Metabolism and Role in Growth (Tryfiates, G. P., ed) pp. 27–51, Food and Nutrition Press, Westport, CT
  8. Schwarts, R., and Kjelgaard, N. O. (1951) Biochem. J. 48, 333–337[Medline] [Order article via Infotrieve]
  9. Stanulovic, M., Jeremic, V., Leskovas, V., and Chaykin, S. (1976) Enzyme 21, 357–369[Medline] [Order article via Infotrieve]
  10. Snell, E. E., and Haskell, B. E. (1971) in Comprehensive Biochemistry (Florkin, M., and Stotz, E. H., eds) Vol. 21, pp. 47–71, Elsevier Scientific Publishing Co., Amsterdam
  11. Anderson, B. B., Newmark, P. A., and Rawlins, M. (1974) Nature 250, 502–504[CrossRef][Medline] [Order article via Infotrieve]
  12. McCormick, D. B., and Merrill, A. H. (1980) in Vitamin B6 Metabolism and Role in Growth (Tryfiates, G. P., ed) pp. 1–26, Food and Nutrition Press, Westport, CT
  13. Kwon, O.-S., Kwok, F., and Churchich, J. E. (1991) J. Biol. Chem. 266, 22136–22140[Abstract/Free Full Text]
  14. Li, T. K., Lumeng, L., and Veitch, R. L. (1974) Biochem. Biophys. Res. Commun. 61, 677–684[CrossRef][Medline] [Order article via Infotrieve]
  15. Lumeng, L., and Li, T. K. (1975) J. Biol. Chem. 250, 8126–8131[Abstract/Free Full Text]
  16. Fonda, M. L. (1992) J. Biol. Chem. 267, 15978–15983[Abstract/Free Full Text]
  17. Harris, H. (1990) Clin. Chim. Acta 186, 133–150[CrossRef][Medline] [Order article via Infotrieve]
  18. Bull, H., Murray, P. G., Thomas, A. M., and Nelson, P. N. (2002) Mol. Pathol. 55, 65–72[Abstract/Free Full Text]
  19. Fonda, M. L., and Zhang, Y. (1995) Arch. Biochem. Biophys. 320, 345–352[Medline] [Order article via Infotrieve]
  20. Gao, G., and Fonda, M. L. (1994) J. Biol. Chem. 269, 7163–7168[Abstract/Free Full Text]
  21. Gao, G., and Fonda, M. L. (1994) J. Biol. Chem. 269, 8234–8239[Abstract/Free Full Text]
  22. Gao, G., and Fonda, M. L. (1994) Arch. Biochem. Biophys. 313, 166–172[Medline] [Order article via Infotrieve]
  23. Thompson, J. D., Hinggins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673–4680[Abstract/Free Full Text]
  24. Rose, Z. B. (1981) Arch. Biochem. Biophys. 208, 602–609[Medline] [Order article via Infotrieve]
  25. Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349–383[CrossRef][Medline] [Order article via Infotrieve]
  26. Padgett, R. A., Grabowski, P. J., Konarska, M. M., Seiler, S., and Sharp, P. A. (1986) Annu. Rev. Biochem. 55, 1119–1150[CrossRef][Medline] [Order article via Infotrieve]
  27. Gardiner-Garden, M., and Frommer, M. (1987) J. Mol. Biol. 196, 261–282[CrossRef][Medline] [Order article via Infotrieve]
  28. Buss, D. D., Hamm, M. W., Mehansho, H., and Henderson, L. M. (1980) J. Nutr. 110, 1655–1663[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
E. Kuznetsova, M. Proudfoot, C. F. Gonzalez, G. Brown, M. V. Omelchenko, I. Borozan, L. Carmel, Y. I. Wolf, H. Mori, A. V. Savchenko, et al.
Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family
J. Biol. Chem., November 24, 2006; 281(47): 36149 - 36161.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/50/50040    most recent
M309619200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jang, Y. M.
Right arrow Articles by Kwon, O.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jang, Y. M.
Right arrow Articles by Kwon, O.-S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement