Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes.

Pyridox(am)ine 5 ' -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 ' -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in Drosophila alleles, hWT , hR116Q , hD33V , and hR95H , in which the endogenous Drosophila PNPO was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that hR95H had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.

Identification and characterization of the pyridoxal 5'-phosphate allosteric site in Escherichia coli pyridoxine 5'-phosphate oxidase.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5'-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24, and Arg215) that form an "arginine cage" and efficiently trap PLP. The crystal structure of the PNPO-PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate-PLP ternary complex catalytically incompetent. Site-directed mutagenesis studies focused on the arginine cage validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R21L variant).

Molecular characterization of pyridoxine 5'-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy.

Defects of vitamin B6 metabolism are responsible for severe neurological disorders, such as pyridoxamine 5'-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5'-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B6 metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.

Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P).

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli.

In Escherichia coli, the synthesis of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, takes place through the so-called deoxyxylulose 5-phosphate-dependent pathway, whose last step is pyridoxine 5'-phosphate (PNP) oxidation to PLP, catalyzed by the FMN-dependent enzyme PNP oxidase (PNPOx). This enzyme plays a pivotal role in controlling intracellular homeostasis and bioavailability of PLP. PNPOx has been proposed to undergo product inhibition resulting from PLP binding at the active site. PLP has also been reported to bind tightly at a secondary site, apparently without causing PNPOx inhibition. The possible location of this secondary site has been indicated by crystallographic studies as two symmetric surface pockets present on the PNPOx homodimer, but this site has never been verified by other experimental means. Here, we demonstrate, through kinetic measurements, that PLP inhibition is actually of a mixed-type nature and results from binding of this vitamer at an allosteric site. This interpretation was confirmed by the characterization of a mutated PNPOx form, in which substrate binding at the active site is heavily hampered but PLP binding is preserved. Structural and functional connections between the active site and the allosteric site were indicated by equilibrium binding experiments, which revealed different PLP-binding stoichiometries with WT and mutant PNPOx forms. These observations open up new horizons on the mechanisms that regulate E. coli PNPOx, which may have commonalities with the mechanisms regulating human PNPOx, whose crucial role in vitamin B6 metabolism and epilepsy is well-known.

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway.

The vitamin B6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup.

PdxH proteins of mycobacteria are typical members of the classical pyridoxine/pyridoxamine 5'-phosphate oxidase family.

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of Mycobacterium tuberculosis (Mtb). PLP functions as a cofactor for 58 putative PLP-binding proteins encoded by the Mtb genome and could also act as a potential antioxidant. De novo biosynthesis of PLP in Mtb takes place through the 'deoxyxylulose 5'-phosphate (DXP)-independent' pathway, whereas PdxH enzymes, possessing pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPOx) activity, are involved in the PLP salvage pathway. In this study, we demonstrate that the annotated PdxH enzymes from various mycobacterial species are bona fide members of the classical PNPOx enzyme family, capable of producing PLP using both pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) substrates.

Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

Analysis of factors influencing hydration site prediction based on molecular dynamics simulations.

Water contributes significantly to the binding of small molecules to proteins in biochemical systems. Molecular dynamics (MD) simulation based programs such as WaterMap and WATsite have been used to probe the locations and thermodynamic properties of hydration sites at the surface or in the binding site of proteins generating important information for structure-based drug design. However, questions associated with the influence of the simulation protocol on hydration site analysis remain. In this study, we use WATsite to investigate the influence of factors such as simulation length and variations in initial protein conformations on hydration site prediction. We find that 4 ns MD simulation is appropriate to obtain a reliable prediction of the locations and thermodynamic properties of hydration sites. In addition, hydration site prediction can be largely affected by the initial protein conformations used for MD simulations. Here, we provide a first quantification of this effect and further indicate that similar conformations of binding site residues (RMSD < 0.5 Å) are required to obtain consistent hydration site predictions.

Plants utilize a highly conserved system for repair of NADH and NADPH hydrates.

NADH and NADPH undergo spontaneous and enzymatic reactions that produce R and S forms of NAD(P)H hydrates [NAD(P)HX], which are not electron donors and inhibit various dehydrogenases. In bacteria, yeast (Saccharomyces cerevisiae), and mammals, these hydrates are repaired by the tandem action of an ADP- or ATP-dependent dehydratase that converts (S)-NAD(P)HX to NAD(P)H and an epimerase that facilitates interconversion of the R and S forms. Plants have homologs of both enzymes, the epimerase homolog being fused to the vitamin B6 salvage enzyme pyridoxine 5'-phosphate oxidase. Recombinant maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) NAD(P)HX dehydratases (GRMZM5G840928, At5g19150) were able to reconvert (S)-NAD(P)HX to NAD(P)H in an ATP-dependent manner. Recombinant maize and Arabidopsis epimerases (GRMZM2G061988, At5g49970) rapidly interconverted (R)- and (S)-NAD(P)HX, as did a truncated form of the Arabidopsis epimerase lacking the pyridoxine 5'-phosphate oxidase domain. All plant NAD(P)HX dehydratase and epimerase sequences examined had predicted organellar targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays confirmed that both start sites were used. Dual import assays with purified pea (Pisum sativum) chloroplasts and mitochondria, and subcellular localization of GFP fusion constructs in tobacco (Nicotiana tabacum) suspension cells, indicated mitochondrial, plastidial, and cytosolic localization of the Arabidopsis epimerase and dehydratase. Ablation of the Arabidopsis dehydratase gene raised seedling levels of all NADHX forms by 20- to 40-fold, and levels of one NADPHX form by 10- to 30-fold. We conclude that plants have a canonical two-enzyme NAD(P)HX repair system that is directed to three subcellular compartments via the use of alternative translation start sites.

Vitamin B(6) salvage enzymes: mechanism, structure and regulation.

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Molecular basis of reduced pyridoxine 5'-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder.

Mutations in pyridoxine 5'-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5'-phosphate oxidase catalyzes the oxidation of pyridoxine 5'-phosphate to pyridoxal 5'-phosphate, the active cofactor form of vitamin B(6) required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is approximately 850-fold less efficient than the wild-type enzyme due to an approximately 192-fold decrease in pyridoxine 5'-phosphate affinity and an approximately 4.5-fold decrease in catalytic activity. There is also an approximately 50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 A crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a beta-strand-loop-beta-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5'-phosphate oxidase mutations.

Cloning and characterization of a pyridoxine 5'-phosphate oxidase from silkworm, Bombyx mori.

A cDNA encoding Pyridoxine 5'-phosphate oxidase (PNPO) from Bombyx mori was cloned and characterized (GenBank accession number: DQ452398). The cDNA encodes a polypeptide of 257 amino acid residues. The recombinant enzyme purified from Escherichia coli exhibited maximal activity at pH 9.0, and the K(m) values for the substrates of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate were determined as 0.65 and 1.15 micromol/l. It was found that B. mori PNPO shares 51.44% homology with humans, but several function-related, key amino acid residues in B. mori PNPO are different from the human and E. Coli gene. B. mori has a single copy of the PNPO gene, which spans a 3.5 kb region and contains five exons and four introns. B. mori PNPO is a homodimer, with each monomer containing nine antiparallel beta-strands and five alpha-helical segments. The secondary structure was deduced from computational study.

Cloning, expression, purification, crystallization and preliminary X-ray studies of a pyridoxine 5'-phosphate oxidase from Mycobacterium smegmatis.

Pyridoxine 5'-phosphate oxidases (PNPOxs) are known to catalyse the terminal step in pyridoxal 5'-phosphate biosynthesis in a flavin mononucleotide-dependent manner in humans and Escherichia coli. Recent reports of a putative PNPOx from Mycobacterium tuberculosis, Rv1155, suggest that the cofactor or catalytic mechanism may differ in Mycobacterium species. To investigate this, a putative PNPOx from M. smegmatis, Msmeg_3380, has been cloned. This enzyme has been recombinantly expressed in E. coli and purified to homogeneity. Good-quality crystals of selenomethionine-substituted Msmeg_3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 A using synchrotron radiation.

The molecular structure of Rv2074, a probable pyridoxine 5'-phosphate oxidase from Mycobacterium tuberculosis, at 1.6 angstroms resolution.

The crystal structure of a conserved hypothetical protein corresponding to open reading frame Rv2074 from Mycobacterium tuberculosis (Mtb) has been solved by the two-wavelength anomalous dispersion method. Refinement of the molecular structure at 1.6 angstroms resolution resulted in an R(work) of 0.178 and an R(free) of 0.204. The crystal asymmetric unit contains an Rv2074 monomer; however, the crystallographic twofold symmetry operation of space group P4(3)2(1)2 generates dimeric Rv2074. Each monomer folds into a six-stranded antiparallel beta-barrel flanked by two alpha-helices. The three-dimensional structure of Rv2074 is very similar to that of Mtb Rv1155, a probable pyridoxine 5'-phosphate oxidase (PNPOx), which corroborates well with the relatively high sequence similarity (52%) between the two. A structural comparison between Rv2074 and Rv1155 revealed that the core structure (a six-stranded beta-barrel) is also well conserved; the major differences between the two lie in the N- and C-termini and in the small helical domain. Two citric acid molecules were observed in the active site of Rv2074, the crystals of which were grown in 0.2 M sodium citrate buffer pH 5.0. The citric acid molecules are bound to Rv2074 by hydrogen-bonding interactions with Thr55, Gln60 and Lys61. One of the two citric acid molecules occupies the same spatial position that corresponds to the position of the phosphate and ribose sugar moieties of the flavin mononucleotide (FMN) in the Mtb Rv1155-FMN, Escherichia coli PNPOx-FMN and human PNPOx-FMN complex structures. Owing to its extensive structural similarity with Mtb Rv1155 and to the E. coli and human PNPOx enzymes, Rv2074 may be involved in the final step in the biosynthesis of pyridoxal 5'-phosphate (PLP; a vitamin B6).

Crystal structure of a putative pyridoxine 5'-phosphate oxidase (Rv2607) from Mycobacterium tuberculosis.

The three-dimensional structure of Rv2607, a putative pyridoxine 5'-phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X-ray crystallography to 2.5 A resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family.

Structures of Mycobacterium tuberculosispyridoxine 5'-phosphate oxidase and its complexes with flavin mononucleotide and pyridoxal 5'-phosphate.

The X-ray crystal structure of a conserved hypothetical protein of molecular weight 16.3 kDa from Mycobacterium tuberculosis corresponding to open reading frame (ORF) Rv1155 has been solved by the multiwavelength anomalous dispersion method and refined at 1.8 A resolution. The crystal structure revealed that Rv1155 is a dimer in the crystal and that each monomer folds into a large and a small domain; the large domain is a six-stranded antiparallel beta-barrel flanked by two small alpha-helices and the small domain is a helix-loop-helix motif. The dimer interface is formed by residues protruding primarily from five of the six beta-strands in each subunit. Based on structural similarity and on ligand binding, it has been established that Rv1155 is a pyridoxine 5'-phosphate oxidase, the Escherichia coli and human counterparts of which catalyse the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino-acid metabolism. The structures of flavin mononucleotide (FMN) and pyridoxal 5'-phosphate (PLP) bound separately to Rv1155 have been determined at 2.2 and 1.7 A resolution, respectively. Only one monomer binds non-covalently to one FMN molecule or to one PLP molecule. Arg55 and Lys57 are the key residues making hydrogen bonds and ionic interactions with the phosphate and ribose groups of the FMN molecule, whereas Arg55 and Arg129 provide hydrogen bonds and ionic interactions with the phosphate group of the PLP. Structural comparisons of Rv1155 from M. tuberculosis with its E. coli and human counterparts demonstrate that the core structure is highly conserved and the FMN-binding site is similarly disposed in each of the structures.

Age-dependent changes of pyridoxal phosphate synthesizing enzymes immunoreactivities and activities in the gerbil hippocampal CA1 region.

In the present study, age-related changes of pyridoxal 5'-phosphate (PLP) synthesizing enzymes, pyridoxal kinase (PLK) and pyridoxine 5'-phosphate oxidase (PNPO), their protein contents and activities were examined in the gerbil hippocampus proper. Significant age-dependent changes in PLK and PNPO immunoreactivities were found in the CA1 region, but not in the CA2/3 region. In the postnatal month 1 (PM 1) group, PLK and PNPO immunoreactivities were detected mainly in the stratum pyramidale of the CA1 region. PLK and PNPO immunoreactivities and their protein contents were highest in the PM 6 group, showing that many CA1 pyramidal cells had strong PLK and PNPO immunoreactivities. Thereafter, PLK and PNPO immunoreactivities started to decrease and were very low at PM 24. Alterations in the change patterns in protein contents and total activities of PLK and PNPO corresponded to the immunohistochemical data, but their specific activities were not altered in any experimental group. Based on double immunofluorescence study, PLK and PNPO immunoreactive cells in the strata oriens and radiatum were identified as GABAergic cells. Therefore, decreases of PLK and PNPO in the hippocampal CA1 region of aged brains may be involved in aging processes related with gamma-aminobutyric acid (GABA) function.

Structure of Escherichia coli pyridoxine 5'-phosphate oxidase in a tetragonal crystal form: insights into the mechanistic pathway of the enzyme.

Escherichia coli pyridoxine 5'-phosphate oxidase (ePNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP) by the FMN oxidation of pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming FMNH(2) and H(2)O(2). The crystal structure of ePNPOx is reported in a tetragonal unit cell at 2.6 A resolution. The three-dimensional fold of this structure is very similar to those of the E. coli and human enzymes that crystallized in trigonal and monoclinic unit cells. However, unlike the previous structures, the tetragonal structure shows major disorder in one of the two subunit domains that has opened up both the active site and a putative tunnel. Comparison of these structures gives an insight into the mechanistic pathway of PNPOx: from the resting enzyme with no substrate bound, to the initial binding of the substrate at the active site, to the catalytic stage and to the release of the catalytic product from the active site.

Structure of the phenazine biosynthesis enzyme PhzG.

PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.