{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T04:13:33Z","timestamp":1785730413871,"version":"3.56.0"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2020,8,12]],"date-time":"2020-08-12T00:00:00Z","timestamp":1597190400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2020,8,12]],"date-time":"2020-08-12T00:00:00Z","timestamp":1597190400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100004271","name":"Sapienza Universit\u00e0 di Roma","doi-asserted-by":"publisher","award":["Progetto di Ateneo"],"award-info":[{"award-number":["Progetto di Ateneo"]}],"id":[{"id":"10.13039\/501100004271","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Istituto Pasteur Italia - Fondazione Cenci Bolognetti"},{"DOI":"10.13039\/501100004462","name":"Consiglio Nazionale delle Ricerche, Italy","doi-asserted-by":"crossref","award":["Italy-Russia bilateral project"],"award-info":[{"award-number":["Italy-Russia bilateral project"]}],"id":[{"id":"10.13039\/501100004462","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Defects of vitamin B<jats:sub>6<\/jats:sub> metabolism are responsible for severe neurological disorders, such as pyridoxamine 5\u2032-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\u2032-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 B<jats:sub>6<\/jats:sub> 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.<\/jats:p>","DOI":"10.1038\/s41598-020-70598-7","type":"journal-article","created":{"date-parts":[[2020,8,12]],"date-time":"2020-08-12T10:06:55Z","timestamp":1597226815000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["Molecular characterization of pyridoxine 5\u2032-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy"],"prefix":"10.1038","volume":"10","author":[{"given":"Anna","family":"Barile","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabel","family":"Nogu\u00e9s","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martino L.","family":"di Salvo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victoria","family":"Bunik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberto","family":"Contestabile","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angela","family":"Tramonti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,8,12]]},"reference":[{"key":"70598_CR1","doi-asserted-by":"publisher","first-page":"850","DOI":"10.1038\/sj.embor.embor914","volume":"4","author":"R Percudani","year":"2020","unstructured":"Percudani, R. & Peracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep.4, 850\u2013854. https:\/\/doi.org\/10.1038\/sj.embor.embor914 (2020).","journal-title":"EMBO Rep."},{"key":"70598_CR2","unstructured":"Mills, P. B., Footitt, E. J., & Clayton, P. T. Vitamin B6 Metabolism and Inborn Errors, in The Online Metabolic and Molecular Bases of Inherited Disease (ed Valle, D.) (2012)."},{"key":"70598_CR3","doi-asserted-by":"publisher","first-page":"1597","DOI":"10.1016\/j.bbapap.2010.12.006","volume":"1814","author":"ML di Salvo","year":"2011","unstructured":"di Salvo, M. L., Contestabile, R. & Safo, M. K. Vitamin B6 salvage enzymes: Mechanism, structure and regulation. Biochim. Biophys. Acta1814, 1597\u20131608. https:\/\/doi.org\/10.1016\/j.bbapap.2010.12.006 (2011).","journal-title":"Biochim. Biophys. Acta"},{"key":"70598_CR4","doi-asserted-by":"publisher","first-page":"385","DOI":"10.1006\/jmbi.2001.5254","volume":"315","author":"ML di Salvo","year":"2002","unstructured":"di Salvo, M. L. et al. Active site structure and stereospecificity of Escherichia coli pyridoxine-5\u2019-phosphate oxidase. J. Mol. Biol.315, 385\u2013397. https:\/\/doi.org\/10.1006\/jmbi.2001.5254 (2002).","journal-title":"J. Mol. Biol."},{"key":"70598_CR5","doi-asserted-by":"publisher","first-page":"2452","DOI":"10.1111\/j.1432-1033.2004.04175.x","volume":"271","author":"JH Kang","year":"2004","unstructured":"Kang, J. H. et al. Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5\u2019-phosphate oxidase. Eur. J. Biochem.271, 2452\u20132461. https:\/\/doi.org\/10.1111\/j.1432-1033.2004.04175.x (2004).","journal-title":"Eur. J. Biochem."},{"key":"70598_CR6","doi-asserted-by":"publisher","first-page":"1455","DOI":"10.1110\/ps.0356203","volume":"12","author":"FN Musayev","year":"2003","unstructured":"Musayev, F. N., Di Salvo, M. L., Ko, T. P., Schirch, V. & Safo, M. K. Structure and properties of recombinant human pyridoxine 5\u2019-phosphate oxidase. Protein Sci.12, 1455\u20131463. https:\/\/doi.org\/10.1110\/ps.0356203 (2003).","journal-title":"Protein Sci."},{"key":"70598_CR7","doi-asserted-by":"publisher","first-page":"15593","DOI":"10.1074\/jbc.RA119.009697","volume":"294","author":"A Barile","year":"2019","unstructured":"Barile, A. et al. Allosteric feedback inhibition of pyridoxine 5\u2019-phosphate oxidase from. J. Biol. Chem.294, 15593\u201315603. https:\/\/doi.org\/10.1074\/jbc.RA119.009697 (2019).","journal-title":"J. Biol. Chem."},{"key":"70598_CR8","doi-asserted-by":"publisher","first-page":"398","DOI":"10.1002\/2211-5463.12042","volume":"6","author":"MS Ghatge","year":"2016","unstructured":"Ghatge, M. S. et al. Inactive mutants of human pyridoxine 5\u2019-phosphate oxidase: A possible role for a noncatalytic pyridoxal 5\u2019-phosphate tight binding site. FEBS Open Biol.6, 398\u2013408. https:\/\/doi.org\/10.1002\/2211-5463.12042 (2016).","journal-title":"FEBS Open Biol."},{"key":"70598_CR9","first-page":"897","volume":"4","author":"ML di Salvo","year":"2012","unstructured":"di Salvo, M. L., Safo, M. K. & Contestabile, R. Biomedical aspects of pyridoxal 5\u2019-phosphate availability. Front. Biosci. Elite Ed4, 897\u2013913 (2012).","journal-title":"Front. Biosci. Elite Ed"},{"key":"70598_CR10","doi-asserted-by":"publisher","first-page":"1614","DOI":"10.1016\/S0140-6736(03)13312-0","volume":"361","author":"PT Clayton","year":"2003","unstructured":"Clayton, P. T., Surtees, R. A., DeVile, C., Hyland, K. & Heales, S. J. Neonatal epileptic encephalopathy. Lancet361, 1614 (2003).","journal-title":"Lancet"},{"key":"70598_CR11","doi-asserted-by":"publisher","first-page":"629","DOI":"10.1002\/jimd.12060","volume":"42","author":"MP Wilson","year":"2019","unstructured":"Wilson, M. P., Plecko, B., Mills, P. B. & Clayton, P. T. Disorders affecting vitamin B. J. Inherit. Metab. Dis.42, 629\u2013646. https:\/\/doi.org\/10.1002\/jimd.12060 (2019).","journal-title":"J. Inherit. Metab. Dis."},{"key":"70598_CR12","doi-asserted-by":"publisher","first-page":"1350","DOI":"10.1093\/brain\/awu051","volume":"137","author":"PB Mills","year":"2014","unstructured":"Mills, P. B. et al. Epilepsy due to PNPO mutations: Genotype, environment and treatment affect presentation and outcome. Brain137, 1350\u20131360. https:\/\/doi.org\/10.1093\/brain\/awu051 (2014).","journal-title":"Brain"},{"key":"70598_CR13","doi-asserted-by":"publisher","first-page":"1077","DOI":"10.1093\/hmg\/ddi120","volume":"14","author":"PB Mills","year":"2005","unstructured":"Mills, P. B. et al. Neonatal epileptic encephalopathy caused by mutations in the PNPO gene encoding pyridox(am)ine 5\u2019-phosphate oxidase. Hum. Mol. Genet.14, 1077\u20131086. https:\/\/doi.org\/10.1093\/hmg\/ddi120 (2005).","journal-title":"Hum. Mol. Genet."},{"key":"70598_CR14","unstructured":"Ghatge MS et al. Molecular defects of vitamin B6 metabolism associated with neonatal epileptic encephalopathy. In Miscellanea on Encephalopathies\u2014A Second Look (2012) Tanasescu R, ed., pp. 267."},{"key":"70598_CR15","doi-asserted-by":"publisher","first-page":"30949","DOI":"10.1074\/jbc.M109.038372","volume":"284","author":"FN Musayev","year":"2009","unstructured":"Musayev, F. N. et al. Molecular basis of reduced pyridoxine 5\u2019-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder. J. Biol. Chem.284, 30949\u201330956. https:\/\/doi.org\/10.1074\/jbc.M109.038372 (2009).","journal-title":"J. Biol. Chem."},{"key":"70598_CR16","doi-asserted-by":"publisher","first-page":"317","DOI":"10.1007\/s10545-005-0243-2","volume":"29","author":"PT Clayton","year":"2006","unstructured":"Clayton, P. T. B6-responsive disorders: A model of vitamin dependency. J. Inherit. Metab. Dis.29, 317\u2013326. https:\/\/doi.org\/10.1007\/s10545-005-0243-2 (2006).","journal-title":"J. Inherit. Metab. Dis."},{"key":"70598_CR17","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1016\/j.ymgme.2008.04.008","volume":"94","author":"M Khayat","year":"2008","unstructured":"Khayat, M. et al. PNPO deficiency: An under diagnosed inborn error of pyridoxine metabolism. Mol. Genet. Metab.94, 431\u2013434. https:\/\/doi.org\/10.1016\/j.ymgme.2008.04.008 (2008).","journal-title":"Mol. Genet. Metab."},{"key":"70598_CR18","doi-asserted-by":"publisher","first-page":"216","DOI":"10.1016\/j.ymgme.2007.10.003","volume":"93","author":"A Ruiz","year":"2008","unstructured":"Ruiz, A. et al. A new fatal case of pyridox(am)ine 5\u2019-phosphate oxidase (PNPO) deficiency. Mol. Genet. Metab.93, 216\u2013218. https:\/\/doi.org\/10.1016\/j.ymgme.2007.10.003 (2008).","journal-title":"Mol. Genet. Metab."},{"key":"70598_CR19","doi-asserted-by":"publisher","first-page":"135","DOI":"10.1016\/j.ymgme.2017.08.003","volume":"122","author":"ML di Salvo","year":"2017","unstructured":"di Salvo, M. L. et al. Pyridoxine-5\u2019-phosphate oxidase (Pnpo) deficiency: Clinical and biochemical alterations associated with the C.347g>A (P..Arg116gln) mutation. Mol. Genet. Metab.122, 135\u2013142. https:\/\/doi.org\/10.1016\/j.ymgme.2017.08.003 (2017).","journal-title":"Mol. Genet. Metab."},{"key":"70598_CR20","doi-asserted-by":"publisher","unstructured":"di Salvo, M. L. et al. Corrigendum to \"Pyridoxine-5'-phosphate oxidase (Pnpo) deficiency: Clinical and biochemical alterations associated with the C.347g>A (P..Arg116gln) mutation\" [Mol. Genet. Metab. 122\/1\u20132 (2017) 135\u2013142]. Mol. Genet. Metab.\nhttps:\/\/doi.org\/10.1016\/j.ymgme.2018.02.017 (2018).","DOI":"10.1016\/j.ymgme.2018.02.017"},{"key":"70598_CR21","doi-asserted-by":"publisher","first-page":"1425","DOI":"10.1212\/WNL.0000000000000344","volume":"82","author":"B Plecko","year":"2014","unstructured":"Plecko, B. et al. Pyridoxine responsiveness in novel mutations of the PNPO gene. Neurology82, 1425\u20131433. https:\/\/doi.org\/10.1212\/WNL.0000000000000344 (2014).","journal-title":"Neurology"},{"key":"70598_CR22","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1016\/j.clineuro.2017.10.019","volume":"163","author":"P Accorsi","year":"2017","unstructured":"Accorsi, P. et al. Pyridoxine responsiveness in pyridox(am)ine-5-phosphate oxidase deficiency: The importance of early treatment. Clin. Neurol. Neurosurg.163, 90\u201393. https:\/\/doi.org\/10.1016\/j.clineuro.2017.10.019 (2017).","journal-title":"Clin. Neurol. Neurosurg."},{"key":"70598_CR23","doi-asserted-by":"publisher","first-page":"498","DOI":"10.1111\/dmcn.12346","volume":"56","author":"TL Ware","year":"2014","unstructured":"Ware, T. L. et al. Typical and atypical phenotypes of PNPO deficiency with elevated CSF and plasma pyridoxamine on treatment. Dev. Med. Child Neurol.56, 498\u2013502. https:\/\/doi.org\/10.1111\/dmcn.12346 (2014).","journal-title":"Dev. Med. Child Neurol."},{"key":"70598_CR24","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1007\/8904_2012_194","volume":"9","author":"PL Pearl","year":"2013","unstructured":"Pearl, P. L. et al. Partial pyridoxine responsiveness in PNPO deficiency. JIMD Rep.9, 139\u2013142. https:\/\/doi.org\/10.1007\/8904_2012_194 (2013).","journal-title":"JIMD Rep."},{"key":"70598_CR25","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1006\/prep.1998.0904","volume":"13","author":"M Di Salvo","year":"1998","unstructured":"di Salvo, M., Yang, E., Zhao, G., Winkler, M. E. & Schirch, V. Expression, purification, and characterization of recombinant Escherichia coli pyridoxine 5\u2019-phosphate oxidase. Protein Exp. Purif.13, 349\u2013356. https:\/\/doi.org\/10.1006\/prep.1998.0904 (1998).","journal-title":"Protein Exp. Purif."},{"key":"70598_CR26","doi-asserted-by":"publisher","first-page":"868","DOI":"10.1016\/j.dib.2017.10.032","volume":"15","author":"ML di Salvo","year":"2017","unstructured":"di Salvo, M. L. et al. Biochemical data from the characterization of a new pathogenic mutation of human pyridoxine-5\u2019-phosphate oxidase (PNPO). Data Brief15, 868\u2013875. https:\/\/doi.org\/10.1016\/j.dib.2017.10.032 (2017).","journal-title":"Data Brief"},{"key":"70598_CR27","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1006\/abbi.2000.1737","volume":"377","author":"ES Yang","year":"2000","unstructured":"Yang, E. S. & Schirch, V. Tight binding of pyridoxal 5\u2019-phosphate to recombinant Escherichia coli pyridoxine 5\u2019-phosphate oxidase. Arch. Biochem. Biophys.377, 109\u2013114. https:\/\/doi.org\/10.1006\/abbi.2000.1737 (2000).","journal-title":"Arch. Biochem. Biophys."},{"key":"70598_CR28","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-7091-1402-5","volume-title":"Kinetics of Enzyme-Modifier Interaction: Selected Topics in the Theory and Diagnosis of Inhibition and Activation Mechanisms","author":"A Baici","year":"2015","unstructured":"Baici, A. Kinetics of Enzyme-Modifier Interaction: Selected Topics in the Theory and Diagnosis of Inhibition and Activation Mechanisms (Springer, Berlin, 2015)."},{"key":"70598_CR29","doi-asserted-by":"publisher","DOI":"10.1111\/febs.15286","author":"C Nardella","year":"2020","unstructured":"Nardella, C. et al. Interaction of Bacillus subtilis GabR with the gabTD promoter: Role of repeated sequences and effect of GABA in transcriptional activation. FEBS J.\u00a0https:\/\/doi.org\/10.1111\/febs.15286 (2020).","journal-title":"FEBS J."},{"key":"70598_CR30","doi-asserted-by":"publisher","first-page":"494","DOI":"10.1016\/j.yebeh.2010.12.046","volume":"20","author":"A Veerapandiyan","year":"2011","unstructured":"Veerapandiyan, A. et al. Electroencephalographic and seizure manifestations of pyridoxal 5\u2019-phosphate-dependent epilepsy. Epilepsy Behav.20, 494\u2013501. https:\/\/doi.org\/10.1016\/j.yebeh.2010.12.046 (2011).","journal-title":"Epilepsy Behav."},{"key":"70598_CR31","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1016\/j.pediatrneurol.2017.05.024","volume":"76","author":"RM Guerriero","year":"2017","unstructured":"Guerriero, R. M. et al. Systemic manifestations in pyridox(am)ine 5\u2019-phosphate oxidase deficiency. Pediatr. Neurol.76, 47\u201353. https:\/\/doi.org\/10.1016\/j.pediatrneurol.2017.05.024 (2017).","journal-title":"Pediatr. Neurol."},{"key":"70598_CR32","doi-asserted-by":"publisher","first-page":"12034","DOI":"10.1021\/bi901568b","volume":"48","author":"M Vivoli","year":"2009","unstructured":"Vivoli, M. et al. Role of a conserved active site cation-pi interaction in Escherichia coli serine hydroxymethyltransferase. Biochemistry48, 12034\u201312046. https:\/\/doi.org\/10.1021\/bi901568b (2009).","journal-title":"Biochemistry"},{"key":"70598_CR33","doi-asserted-by":"publisher","first-page":"885","DOI":"10.1006\/jmbi.2001.5009","volume":"312","author":"AH Elcock","year":"2001","unstructured":"Elcock, A. H. Prediction of functionally important residues based solely on the computed energetics of protein structure. J. Mol. Biol.312, 885\u2013896. https:\/\/doi.org\/10.1006\/jmbi.2001.5009 (2001).","journal-title":"J. Mol. Biol."},{"key":"70598_CR34","volume-title":"Enzyme Kinetics; Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems","author":"IH Segel","year":"1975","unstructured":"Segel, I. H. Enzyme Kinetics; Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems (Wiley, New York, 1975)."},{"key":"70598_CR35","doi-asserted-by":"publisher","first-page":"817","DOI":"10.1006\/jmbi.2001.4734","volume":"310","author":"MK Safo","year":"2001","unstructured":"Safo, M. K., Musayev, F. N., di Salvo, M. L. & Schirch, V. X-ray structure of Escherichia coli pyridoxine 5\u2019-phosphate oxidase complexed with pyridoxal 5\u2019-phosphate at 2.0 A resolution. J. Mol. Biol.310, 817\u2013826. https:\/\/doi.org\/10.1006\/jmbi.2001.4734 (2001).","journal-title":"J. Mol. Biol."},{"key":"70598_CR36","doi-asserted-by":"publisher","first-page":"56","DOI":"10.1186\/1472-6807-7-56","volume":"7","author":"S Khan","year":"2007","unstructured":"Khan, S. & Vihinen, M. Spectrum of disease-causing mutations in protein secondary structures. BMC Struct. Biol.7, 56. https:\/\/doi.org\/10.1186\/1472-6807-7-56 (2007).","journal-title":"BMC Struct. Biol."},{"key":"70598_CR37","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1038\/274775a0","volume":"274","author":"C Coulondre","year":"1978","unstructured":"Coulondre, C., Miller, J. H., Farabaugh, P. J. & Gilbert, W. Molecular basis of base substitution hotspots in Escherichia coli. Nature274, 775\u2013780. https:\/\/doi.org\/10.1038\/274775a0 (1978).","journal-title":"Nature"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-70598-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-70598-7","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-70598-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,6]],"date-time":"2022-12-06T15:29:52Z","timestamp":1670340592000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-70598-7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,8,12]]},"references-count":37,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,12]]}},"alternative-id":["70598"],"URL":"https:\/\/doi.org\/10.1038\/s41598-020-70598-7","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,8,12]]},"assertion":[{"value":"28 May 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"31 July 2020","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"12 August 2020","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"13621"}}