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Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salva...

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Autores principales: Ghatge, Mohini S., Contestabile, Roberto, di Salvo, Martino L., Desai, Jigar V., Gandhi, Amit K., Camara, Christina M., Florio, Rita, González, Isabel N., Parroni, Alessia, Schirch, Verne, Safo, Martin K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404986/
https://www.ncbi.nlm.nih.gov/pubmed/22848564
http://dx.doi.org/10.1371/journal.pone.0041680
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author Ghatge, Mohini S.
Contestabile, Roberto
di Salvo, Martino L.
Desai, Jigar V.
Gandhi, Amit K.
Camara, Christina M.
Florio, Rita
González, Isabel N.
Parroni, Alessia
Schirch, Verne
Safo, Martin K.
author_facet Ghatge, Mohini S.
Contestabile, Roberto
di Salvo, Martino L.
Desai, Jigar V.
Gandhi, Amit K.
Camara, Christina M.
Florio, Rita
González, Isabel N.
Parroni, Alessia
Schirch, Verne
Safo, Martin K.
author_sort Ghatge, Mohini S.
collection PubMed
description Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B(6) proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6) enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
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spelling pubmed-34049862012-07-30 Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase Ghatge, Mohini S. Contestabile, Roberto di Salvo, Martino L. Desai, Jigar V. Gandhi, Amit K. Camara, Christina M. Florio, Rita González, Isabel N. Parroni, Alessia Schirch, Verne Safo, Martin K. PLoS One Research Article Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B(6) proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6) enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell. Public Library of Science 2012-07-25 /pmc/articles/PMC3404986/ /pubmed/22848564 http://dx.doi.org/10.1371/journal.pone.0041680 Text en © 2012 Ghatge et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ghatge, Mohini S.
Contestabile, Roberto
di Salvo, Martino L.
Desai, Jigar V.
Gandhi, Amit K.
Camara, Christina M.
Florio, Rita
González, Isabel N.
Parroni, Alessia
Schirch, Verne
Safo, Martin K.
Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase
title Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase
title_full Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase
title_fullStr Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase
title_full_unstemmed Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase
title_short Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase
title_sort pyridoxal 5′-phosphate is a slow tight binding inhibitor of e. coli pyridoxal kinase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404986/
https://www.ncbi.nlm.nih.gov/pubmed/22848564
http://dx.doi.org/10.1371/journal.pone.0041680
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