Structure and properties of recombinant human pyridoxine 5'-phosphate oxidase

Pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5'-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of approximately 0.2 sec(-1) and K(m) values in the low micromolar range for both pyridoxine 5'phosphate and pyridoxamine 5'-phosphate. Pyridoxal 5'-phosphate is an effective product inhibitor. The three-dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5'-phosphate tightly on each subunit.     

4-Phospho-hydroxy-l-threonine is an obligatory intermediate in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12

Abstract We show that thrB -encoded homoserine kinase is required for growth of Escherichia coli K-12 pdxB mutants on minimal glucose medium supplemented with 4-hydroxy-l-threonine (synonym, 3-hydroxyhomoserine) or d-glycolaldehyde. This result is consistent with a model in which 4-phospho-hydroxy-l-threonine (synonym, 3-hydroxyhomoserine phosphate), rather than 4-hydroxy-l-threonine, is an obligatory intermediate in pyridoxal 5'-phosphate biosynthesis. Ring closure using 4-phospho-hydroxy-l-threonine as a substrate would lead to the formation of pyridoxine 5'-phosphate, and not pyridioxine, as the first B₆-vitamer synthesized de novo. These considerations suggest that E. coli pyridoxal/pyridoxamine/pyridoxine kinase is not required for the main de novo pathway of pyridoxal 5'-phosphate biosynthesis, and instead plays a role only in the B₆-vitamer salvage pathway.     

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay.

An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH. The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5'-phosphate in the original sample. The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5'-phosphate content. Other B6 vitamers are converted to pyridoxal 5'-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5'-phosphate oxidase. A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5'-phosphate. The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli. The results show that the pool of pyridoxal 5'-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5'-phosphate oxidase.     

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution

BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.     

Modification of ribonucleic acid by vitamin B6. 1. Specific interaction of pyridoxal 5'-phosphate with transfer ribonucleic acid.

Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate treated tRNA digest showed that pyridoxal 5'-phosphate binds specifically to GMP, presumably in the form of a Schiff base with the exocyclic amino group of the purine. The reaction of pyridoxal 5'-phosphate with whole tRNA was competitively inhibited by N-acetoxy-2-acetylaminofluorene. This suggests that binding occurred primarily to the G20 base residue at the unpaired region of the dihydrouridine loop (Fujimura et al., 1972). The modification of tRNA by pyridoxal 5'-phosphate resulted in the inhibition, to varying extent (10-80%), of amino acid acceptance in the aminoacyl-tRNA synthetase reaction. Defects in codon recognition by pyridoxal 5'-phosphate modified amino acid acylated tRNAs in the presence of the corresponding guanine-containing polynucleotide triplets were observed by the ribosomal binding assay.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Characterization of pyridoxine auxotrophs of Escherichia coli: serine and pdxF mutants

At least six phenotypically distinct classes of mutants of Escherichia coli which require serine or pyridoxine or both can be isolated. Three of the six classes lack 3-phosphoserine-2-oxoglutarate aminotransferase. One of these classes contains WG5, a mutant previously characterized as containing the pdxF5 allele. The aminotransferase isolated from this mutant has been compared to that isolated from wild-type E. coli and found to have apparently normal affinity for pyridoxal 5'-phosphate, but reduced affinity for pyridoxamine 5'-phosphate.     

Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis.

The tyrS genes from Escherichia coli and Bacillus stearothermophilus were toxic to E. coli when they were carried by plasmids with very high copy numbers (pEMBL8 and pEMBL9). We quantified this effect by comparing the efficiencies of plating of E. coli derivatives harboring recombinant plasmids in various experimental conditions. The toxicity was apparent at both 30 and 37 degrees C. It increased with the growth temperature, the strength of the tyrS promoter, and the copy number of the plasmidic vector. Two- to threefold enhancement of tyrS expression raised the toxicity 300-fold. Point mutations in tyrS that prevent interaction between its product, tyrosyl-tRNA synthetase, and tRNA(Tyr) but do not alter the rate of formation of tyrosyl-adenylate abolished the toxicity. Thus, the toxic effect was due to high cellular levels of synthetase activity. At 30 degrees C, the cellular concentration of tyrosyl-tRNA synthetase reached 55% of that of soluble proteins and led to decreased beta-galactosidase stability. We discuss possible causes of this toxic effect and describe its applications to the study of the recognition and interaction between the synthetase and tRNA(Tyr).     

Isolation of a pdxJ point mutation that bypasses the requirement for the PdxH oxidase in pyridoxal 5' -phosphate coenzyme biosynthesis in Escherichia coli K-12.

We isolated 26 suppressor mutations that allowed growth of a delta pdxH::omega null mutant in the absence of pyridoxal. Each suppressor mapped to pdxJ, and the eight suppressors sequenced contained the same glycine-to-serine change in the PdxJ polypeptide. This bypass suppression suggests that PdxJ may participate in formation of the pyridine ring of pyridoxine 5'-phosphate.     

Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12.

The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation. An nth insertion mutant and a deletion mutant were studied. nth is located between add and tyrS on the linkage map of E. coli K-12 and was 97% linked to tyrS in a transduction with phage P1.     

Synthesis of Pyridoxine by a Pyridoxal Auxotroph of Escherichia coli

Dempsey, Walter B. (University of Florida, Gainesville). Synthesis of pyridoxine by a pyridoxal auxotroph of Escherichia coli. J. Bacteriol. 92:333-337. 1966.-A pyridoxal auxotroph of Escherichia coli B produced pyridoxol and pyridoxol 5'-phosphate during starvation for pyridoxal. The identification of these compounds was made both by bioassay and by ion-exchange chromatography. Pyridoxol 5'-phosphate oxidase activity was absent in extracts of the auxotroph. The rate of synthesis of total pyridoxine by a pyridoxal-starved culture of this auxotroph was 6.0 x 10(-6) moles per mg per hr. Cellular content of pyridoxine was constant at 4.0 x 10(-10) moles/mg.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.     

Identification of amino acid residues modified by pyridoxal 5'-phosphate in Escherichia coli glutamine synthetase

Chemical modification studies with pyridoxal 5'-phosphate have indicated that lysine(s) appear to be at or near the active site of Escherichia coli glutamine synthetase (Colanduoni, J., and Villafranca, J. J. (1985) J. Biol. Chem. 260, 15042-15050; Whitley, E. J., Jr., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). Enzyme samples were prepared that contained approximately 1, approximately 2, and approximately 3 pyridoxamine 5'-phosphate residues/50,000-Da monomer; the activity of each sample was 100, 25, and 14% of the activity of unmodified enzyme, respectively. Cyanogen bromide cleavage of each enzyme sample was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their absorbance at 320 nm. These isolated peptides were analyzed for amino acid composition and sequenced. The N terminus of the protein (a serine residue) was modified by pyridoxal 5'-phosphate at a stoichiometry of approximately 1/50,000 Da and this modified enzyme had full catalytic activity. Beyond a stoichiometry of approximately 1, lysines 383 and 352 reacted with pyridoxal 5'-phosphate and each modification results in a partial loss of activity. When various combinations of substrates and substrate analogs (ADP/Pi or L-methionine-SR-sulfoximine phosphate/ADP) were used to protect the enzyme from modification, Lys-352 was protected from modification indicating that this residue is at the active site. Under all experimental conditions employed, Lys-47, which reacts with the ATP analog 5'-p-fluorosulfonylbenzoyl-adenosine does not react with pyridoxal 5'-phosphate.     

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.     

Mutants of Escherichia coli with a growth requirement for either lysine or pyridoxine

Two distinct phenotypic classes of lysine requiring auxotrophs of Escherichia coli are described. Mutants of the LysA class produce little or no active diaminopimelic acid (DAP) decarboxylase and specifically require lysine for growth. Mutants of the LysB class produce a cryptic DAP decarboxylase which can be activated both in vivo and in vitro by higher than normal levels of its cofactor, pyridoxal 5'-phosphate. The LysB mutants have an alternate requirement for lysine or pyridoxine. Both LysA and LysB mutations map at 55 min, close to the thyA locus of E. coli. The association between pyridoxal phosphate and DAP decarboxylase appears to be much weaker in LysB mutants than in wild-type bacteria, and the mutant enzyme also sediments more slowly than wild-type enzyme in sucrose density gradients. The results suggest that the LysB mutations alter a specific region (or subunit) of the enzyme molecule which is needed to stabilize the binding of pyridoxal phosphate. These studies help to resolve certain contradictory observations on DAP decarboxylase reported earlier and may have relevance to pyridoxal phosphate enzymes in general. Prototrophic revertants of LysB mutants arise by second site mutations that result in increased availability of intracellular pyridoxal phosphate. These revertants appear to be derepressed for pyridoxine biosynthesis.