Pyridoxine 5'-phosphate Oxidase Research Articles

PNPO-PLP axis senses prolonged hypoxia in macrophages by regulating lysosomal activity.

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing systems inducing acute hypoxic responses, including the hypoxia-inducible factor pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here we show that pyridoxine 5'-phosphate oxidase (PNPO), which catalyses bioactivation of vitamin B6, serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decreased PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and inhibited lysosomal acidification, which in macrophages led to iron dysregulation, TET2 protein loss and delayed resolution of the inflammatory response. Among PLP-dependent metabolism, supersulfide synthesis was suppressed in prolonged hypoxia, resulting in the lysosomal inhibition and consequent proinflammatory phenotypes of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing that gradually shuts down PLP-dependent metabolism in response to prolonged oxygen deprivation.

Targeting PNPO to suppress tumor growth via inhibiting autophagic flux and to reverse paclitaxel resistance in ovarian cancer.

Our previous study showed that pyridoxine 5'-phosphate oxidase (PNPO) is a tissue biomarker of ovarian cancer (OC) and has a prognostic implication but detailed mechanisms remain unclear. The current study focused on PNPO-regulated lysosome/autophagy-mediated cellular processes and the potential role of PNPO in chemoresistance. We found that PNPO was overexpressed in OC cells and was a prognostic factor in OC patients. PNPO significantly promoted cell proliferation via the regulation of cyclin B1 and phosphorylated CDK1 and shortened the G2M phase in a cell cycle. Overexpressed PNPO enhanced the biogenesis and perinuclear distribution of lysosomes, promoting the degradation of autophagosomes and boosting the autophagic flux. Further, an autolysosome marker LAMP2 was upregulated in OC cells. Silencing LAMP2 suppressed cell growth and induced cell apoptosis. LAMP2-siRNA blocked PNPO action in OC cells, indicating that the function of PNPO on cellular processes was mediated by LAMP2. These data suggest the existence of the PNPO-LAMP2 axis. Moreover, silencing PNPO suppressed xenographic tumor formation. Chloroquine counteracted the promotion effect of PNPO on autophagic flux and inhibited OC cell survival, facilitating the inhibitory effect of PNPO-shRNA on tumor growth in vivo. Finally, PNPO was overexpressed in paclitaxel-resistant OC cells. PNPO-siRNA enhanced paclitaxel sensitivity in vitro and in vivo. In conclusion, PNPO has a regulatory effect on lysosomal biogenesis that in turn promotes autophagic flux, leading to OC cell proliferation, and tumor formation, and is a paclitaxel-resistant factor. These data imply a potential application by targeting PNPO to suppress tumor growth and reverse PTX resistance in OC.

Riboflavin kinase and pyridoxine 5'-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery.

Enzymes catalysing sequential reactions have developed different mechanisms to control the transport and flux of reactants and intermediates along metabolic pathways, which usually involve direct transfer of metabolites from an enzyme to the next one in a cascade reaction. Despite the fact that metabolite or substrate channelling has been widely studied for reactant molecules, such information is seldom available for cofactors in general, and for flavins in particular. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) act as cofactors in flavoproteins and flavoenzymes involved in a wide range of physiologically relevant processes in all type of organisms. Homo sapiens riboflavin kinase (RFK) catalyses the biosynthesis of the flavin mononucleotide cofactor, and might directly interplay with its flavin client apo-proteins prior to the cofactor transfer. Non-etheless, none of such complexes has been characterized at molecular or atomic level so far. Here, we particularly evaluate the interaction of riboflavin kinase with one of its potential FMN clients, pyridoxine-5'-phosphate oxidase (PNPOx). The interaction capacity of both proteins is assessed by using isothermal titration calorimetry, a methodology that allows to determine dissociation constants for interaction in the micromolar range (in agreement with the expected transient nature of the interaction). Moreover, we show that; i) both proteins become thermally stabilized upon mutual interaction, ii) the tightly bound FMN product can be transferred from RFK to the apo-form of PNPOx producing an efficient enzyme, and iii) the presence of the apo-form of PNPOx slightly enhances RFK catalytic efficiency. Finally, we also show a computational study to predict likely RFK-PNPOx binding modes that can envisage coupling between the FMN binding cavities of both proteins for the potential transfer of FMN.

Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.

Thiamine-dependent regulation of mammalian brain pyridoxal kinase in vitro and in vivo.

Vitamins B1 (thiamine) and B6 (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg2+ but activating the Zn2+ -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B1 - and B6 -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in Kapp of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased Kapp of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.

Prognostic Implication and Oncogenic Role of PNPO in Pan-Cancer.

Objective: Pyridoxine 5′-phosphate oxidase (PNPO) is a key enzyme in the metabolism of vitamin B6 and affects the tumorigenesis of ovarian and breast cancers. However, the roles of PNPO in other types of cancer remain unknown. Methods: The expression of PNPO was interpreted by The Cancer Genome Atlas (TCGA) database and Genotype Tissue-Expression (GTEX) database. Analysis of PNPO genomic alterations and protein expression in human organic tissues was analyzed by the cBioPortal database and human multiple organ tissue arrays. PNPO with drug sensitivity analysis was performed from the CellMiner database. The correlations between PNPO expression and survival outcomes, clinical features, DNA mismatch repair system (MMR), microsatellite instability (MSI), tumor mutation burden (TMB), and immune-associated cell infiltration were analyzed using the TCGA, ESTIMATE algorithm, and TIMER databases. Gene Set Enrichment Analysis (GSEA) was applied to elucidate the biological function of PNPO in pan-cancer. Results: The differential analysis showed that the level of PNPO mRNA expression was upregulated in 21 tumor types compared with normal tissues, which was consistent with its protein expression in most cancer types. The abnormal expression of PNPO could predict the survival outcome of patients with esophageal carcinoma (ESCA), kidney renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), ovarian serous cystadenocarcinoma (OV), and uveal melanoma (UVM). Furthermore, the most frequent mutation type of PNPO genomic was amplified. Moreover, the aberrant PNPO expression was related to MMR, MSI, TMB, and drug sensitivity in various types of cancer. The expression of PNPO was related to the infiltration levels of various immune-associated cells in pan-cancer by ESTIMATE algorithm and TIMER database mining. Conclusion: Our results suggest that PNPO is a potential molecular biomarker for predicting patient prognosis, drug sensitivity, and immunoreaction in pan-cancer.

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).

Nephrotoxicity Profile of Cadmium Revealed by Proteomics in Mouse Kidney.

Cadmium (Cd) is a highly toxic metal and kidney is its main target. However, the molecular effects and associated potential impacts of Cd-accumulated kidney have not been well investigated. In this study, mouse was used as a model to investigate the Cd-induced proteomic profile change in kidney, and a total of 34 differentially expressed proteins were detected by two-dimensional gel electrophoresis (2-DE) and further identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Through Gene Ontology analysis and KEGG pathway annotation, it showed that Cd-regulated kidney metabolism and promoted renal damage and cell migration. By validation of Western blotting and RT-qPCR, metastasis-related proteins LIM and SH3 domain protein 1 (LASP1) and phosphoenolpyruvate carboxykinase/cytosolic [GTP] (PEPCK1) were confirmed to be upregulated; Acyl-CoA synthetase medium-chain family member 3 (ACSM3) was downregulated. Furthermore, carcinoma development-related proteins initiation factor 4A (eIF4A) and pyridoxine-5'-phosphate oxidase (PNPO) were upregulated, and pyridoxal kinase (PK) was downregulated. The downregulation of Na(+)/H(+) exchange regulatory cofactor (NHERF3) might promote renal damage which associated with decrease of transferrin (TRF) in kidney. Taken together, our results revealed proteomic profile of Cd-induced nephrotoxicity and provided data for further insights into the mechanisms of Cd toxicity.

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.

Pyridoxamine Supplementation Effectively Reverses the Abnormal Phenotypes of Zebrafish Larvae With PNPO Deficiency.

Neonatal epileptic encephalopathy (NEE), as a result of pyridoxine 5′-phosphate oxidase (PNPO) deficiency, is a rare neural disorder characterized by intractable seizures and usually leads to early infant death. The clinical phenotypes do not respond to antiepileptic drugs but are alleviated in most cases by giving large doses of pyridoxal 5′-phosphate (PLP). PLP is the active form of vitamin B6 participating in more than 100 enzymatic pathways. One of the causes of NEE is pathogenic mutations in the gene for human PNPO (hPNPO). PNPO is a key enzyme in converting pyridoxine (PN), the common dietary form of vitamin B6, and some other B6 vitamers to PLP. More than 25 different mutations in hPNPO, which result in reduced catalytic activity, have been described for PNPO-deficiency NEE. To date, no animal model is available to test new therapeutic strategies. In this report, we describe using zebrafish with reduced activity of Pnpo as an animal model. Knocking down zPnpo resulted in developmental anomalies including brain malformation and impaired locomotor activity, similar to the clinical features of PNPO-deficiency NEE. Other anomalies include a defective circulation system. These anomalies were significantly alleviated by co-injecting either zpnpo or hPNPO mRNAs. As expected from clinical observations in humans, supplementing with PLP improved the morphological and behavioral anomalies. PN only showed marginal positive effects, and only in a few anomalies. Remarkably, pyridoxamine (PM), another dietary form of vitamin B6, showed rescue effects even at a lower concentration than PLP, presenting a possible new therapeutic treatment for PNPO-deficiency NEE. Finally, GABA, a neurotransmitter whose biosynthesis depends on a PLP-dependent enzyme, showed some positive rescue effect. These results suggest zebrafish to be a promising PNPO-deficiency model for studying PLP homeostasis and drug therapy in vivo.

Gene cloning and comparative analysis of pyridoxal 5′-phosphate salvage synthesis enzymes in tobacco plants

Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an important cofactor for many biochemical transformations. Plants are able to synthesize PLP de novo, but they also have a salvage pathway that functions to convert different vitamer forms between each other. Although the salvage enzymes are identified successively from plants, many questions remain unanswered. In the salvage pathway, PLP is synthesized by an ATP-dependent pyridoxal kinase (PLK) and an FMN-dependent pyridoxine 5′-phosphate oxidase (PNPO). In this study, cDNAs encoding PLK and PNPO were cloned from tobacco plants, compared and analyzed, and then the gene expression was down-regulated by RNA interference. Our results show that the NtPLK and NtPNPO contains highly conserved motifs involved in substrate binding or catalysis. NtPLK and NtPNPO are essential enzymes for all tissues of tobacco plants, and NtPNPO is mainly located in chloroplast. The down-regulation of NtPLK and NtPNPO has a greater impact on the transcription level of other vitamin B6 metabolic enzymes. Based on the results, we speculate that, in plants, the de novo synthesized free PLP may be hydrolyzed to PL by phosphatase in cytosol, and then the PL enter into organelles. In organelles, salvage pathway plays its role to produce PLP for maintenance of vitamin B6-mediated processes.

Pyridoxine 5'-phosphate oxidase is correlated with human breast invasive ductal carcinoma development.

Pyridoxine 5′-phosphate oxidase (PNPO) is a converting enzyme for an active form of vitamin B6. This study aims to evaluate the biological function and the regulatory mechanism of PNPO in human breast invasive ductal carcinoma (IDC). We unveiled for the first time that PNPO was upregulated in patients with IDC and was correlated with the overall survival of patients with metastasis at the later stages. Suppression of PNPO inhibited breast cancer cell proliferation, migration, invasion and colony formation, arrested cell cycle at the G2/M phase and induced cell apoptosis. PNPO was positively correlated with lncRNA MALAT1 which was negatively correlated with miR-216b-5p. PNPO was down-regulated and up-regulated by miR-216b-5p mimics and inhibitors, respectively, in breast cancer cells. A microRNA response element was found in both PNPO and MALAT1 transcripts for miR-216b-5p and the dual-luciferase reporter assay confirmed the binding of these transcripts. Knockdown of MALAT1 resulted in an increase of miR-216b-5p and a decrease of PNPO mRNA, indicating a regulatory mechanism of competing endogenous RNAs. Taken together, these results reveal the biological function and a regulatory mechanism of PNPO, in which the MALAT1/miR-216b-5p/PNPO axis may be important in IDC development. Targeting this axis may have therapeutic potential for breast cancer.

Functional identification of the proximal promoter region of human pyridoxine 5′-phosphate oxidase gene

Pyridoxine 5′-phosphate oxidase (PNPO) is a key enzyme for the biosynthesis of pyridoxal 5′-phosphate. Pyridoxal 5′-phosphate is the catalytically active form of vitamin B6, and acts as a cofactor involved in a high diversity of biochemical reactions. The expression and regulation of PNPO are implicated in numerous physiological and pathological processes. The genomic organization of human PNPO has been reported previously whereas the promoter functional identification is not there yet. In this study, we identified the proximal promoter region of human PNPO gene and analyzed its function. Bioinformatics analysis showed that the transcription start site is at 153 bp upstream of the translation initiation site ATG. Progressive truncation analysis of the 5′-flanking region of PNPO gene demonstrated that two important regulatory regions are located at −996/−852 and −412/+85 bp relative to the transcription start site. From the regulatory regions one USF site, two E2F1 site and multiple Sp1 sites were found. Deletion and mutation experiments indicated that these cis-regulatory elements contribute to the basic promoter activity on different degrees. Downregulation of the transcription factors further indicated that E2F1 plays a dominant role in the transcriptional regulation of PNPO through the binding site at −867/−857, which was confirmed by ChIP assay. Our present study should facilitate further studies on the mechanism regulating the expression of this important gene.

Pyridoxine 5\u2032-phosphate oxidase is a novel therapeutic target and regulated by the TGF-\u03b2 signalling pathway in epithelial ovarian cancer

Pyridoxine 5′-phosphate oxidase (PNPO) is an enzyme that converts pyridoxine 5′-phosphate into pyridoxal 5′-phosphate (PLP), an active form of vitamin B6 implicated in several types of cancer. However, the role of PNPO and its regulatory mechanism in epithelial ovarian cancer (EOC) are unknown. In the present study, PNPO expression in human ovarian tumour tissue and its association with the clinicopathological features of patients with EOC were examined. Further, the biological function of PNPO in EOC cells and in xenograft was evaluated. We demonstrated for the first time that PNPO was overexpressed in human EOC. Knockdown of PNPO induced EOC cell apoptosis, arrested cell cycle at G2/M phase, decreased cell proliferation, migration and invasion. Xenografts of PNPO-shRNA-expressing cells into the nude mouse attenuated tumour growth. PNPO at mRNA and protein levels in EOC cells was decreased after transforming growth factor-β1 (TGF-β1) treatment. The inhibitory effect of TGF-β1 on PNPO expression was abolished in the presence of SB-431542, a TGF-β type I receptor kinase inhibitor. Moreover, we found that TGF-β1-mediated PNPO expression was at least in part through the upregulation of miR-143-3p. These data indicate a mechanism underlying PNPO regulation by the TGF-β signalling pathway. Furthermore, PLP administration reduced PNPO expression and decreased EOC cell proliferation, suggesting a feedback loop between PLP and PNPO. Thus, our findings reveal that PNPO can serve as a novel tissue biomarker of EOC and may be a potential target for therapeutic intervention.

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.

Inactive mutants of human pyridoxine 5'-phosphate oxidase: a possible role for a noncatalytic pyridoxal 5'-phosphate tight binding site.

Pyridoxal 5′‐phosphate (PLP) is a cofactor for many vitamin B6‐requiring enzymes that are important for the synthesis of neurotransmitters. Pyridoxine 5′‐phosphate oxidase (PNPO) is one of two enzymes that produce PLP. Some 16 known mutations in human PNPO (hPNPO), including R95C and R229W, lead to deficiency of PLP in the cell and have been shown to cause neonatal epileptic encephalopathy (NEE). This disorder has no effective treatment, and is often fatal unless treated with PLP. In this study, we show that R95C hPNPO exhibits a 15‐fold reduction in affinity for the FMN cofactor, a 71‐fold decrease in affinity for the substrate PNP, a 4.9‐fold decrease in specific activity, and a 343‐fold reduction in catalytic activity, compared to the wild‐type enzyme. We have reported similar findings for R229W hPNPO. This report also shows that wild‐type, R95C and R229W hPNPO bind PLP tightly at a noncatalytic site and transfer it to activate an apo‐B6 enzyme into the catalytically active holo‐form. We also show for the first time that hPNPO forms specific interactions with several B6 enzymes with dissociation constants ranging from 0.3 to 12.3 μm. Our results suggest a possible in vivo role for the tight binding of PLP in hPNPO, whether wild‐type or variant, by protecting the very reactive PLP, and transferring this PLP directly to activate apo‐B6 enzymes.

Molecular Docking Studies of PA-824 with Pyridoxine 5'-Phosphate Oxidase

Mycobacterium tuberculosis is to be one of the world’s most deadly pathogens. The PA-824 nitroimidazole is a prodrug with an action activity against both hypoxic and replicating of Mycobacterium tuberculosis. PA-824 molecule was activated by a cofactor, which is a compound that binds to a protein. This prodrug was used as a ligand to bind with the protein PDB ID: 1XXO (Pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate). Pyridoxine 5-phosphate oxidase is called as the enzyme that catalyzes the B6 metabolism pathway by limits the rate step process. Docking analysis was performed on the protein 1XX0 along with ligand PA-824 using Autodock.

Rv2607 from Mycobacterium tuberculosis Is a Pyridoxine 5′-Phosphate Oxidase with Unusual Substrate Specificity

Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5′-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5′-phosphate (PNP) or pyridoxamine 5′-phosphate (PMP) to form pyridoxal 5′-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The kcat and KM for this reaction were 0.01 s−1 and 360 µM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.

Crystal structure of the novel PaiB transcriptional regulator from Geobacillus stearothermophilus

The pai operon is transcriptionally regulated by PaiB and has been found to be essential for cell growth and involved in the negative control of sporulation and degradative enzyme production. The pai operon was initially cloned as a fragment of the Bacillus subtilis chromosome consisting of two genes paiA and paiB.1 The presence of both of these genes on a multicopy vector revealed that both decrease the levels of extracellular degradative enzymes such as subtilisin, neutral protease, levansucrase, α-amylase, and alkaline phosphatases and also greatly reduce the frequency of sporulation.2 Based on the study by Strauch (1993), paiB appears to encode a regulator belonging to the same family as the AbrB, Hpr, and Sin regulators. AbrB, Hpr, Sin, and PaiB control the synthesis of degradative enzymes that occurs during the transition between vegetative growth and the onset of the stationary phase and sporulation in B. subtilis.2 Known sporulation genes have been identified as targets for direct control by AbrB (spo0E, spo0H, and spoVG) and Sin (spoIIA, spoIID, spoIIE, and spoIIG).3 Hpr and PaiB may also regulate spo genes directly as evidenced by the Spo phenotype of strains carrying multicopy plasmids that overexpress these two genes.1 Preliminary bioinformatic data suggested that PaiB might be a split barrel flavin-binding regulator. Herein, we report the first crystal structure of the Pai transcriptional regulator: PaiB from Geobacillus stearothermophilus. The structure of PaiB demonstrates that it is a homodimer and that each monomer consists of three domains: large, intermediate, and C-terminal. The large and intermediate domains fold into a structure similar to pyridoxine 5′-phosphate oxidase (PNPO) and form an analogous flavin mononucleotide (FMN)-binding cavity. The small C-terminal domain of PaiB possesses a helix-turn-helix (HTH) DNA-binding fold. The comparisons of sequence and structure show that PaiB is a representative of a new sequence family of transcriptional regulators. Detailed analysis of the protein structure provides insights into the potential biological function.

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.