title: On the mechanism of Escherichia coli pyridoxal kinase inhibition by pyridoxal and pyridoxal 5'-phosphate.
abstract: Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
authors: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors: Martino Luigi
authors: ML
authors: di Salvo
authors: Istituto di Biologia Ambientale e Forestale, Consiglio Nazionale delle Ricerche, Via Salaria Km 29.300, 00015 Monterotondo Scalo, Roma, Italy.
authors: Isabel
authors: I
authors: Nogués
authors: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors: Alessia
authors: A
authors: Parroni
authors: Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Piazzale Aldo Moro 5, 00185 Roma, Italy; Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors: Angela
authors: A
authors: Tramonti
authors: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors: Teresa
authors: T
authors: Milano
authors: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
authors: Stefano
authors: S
authors: Pascarella
authors: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy. Electronic address: roberto.contestabile@uniroma1.it.
authors: Roberto
authors: R
authors: Contestabile
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D011730:Pyridoxal / Q000494:pharmacology*; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*
chemical_list: D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D011731:Pyridoxal Kinase
keywords: Carbinolamine intermediate; Catalytic mechanism; Enzyme regulation; Inhibition mechanism; Pyridoxal phosphate; Vitamin B(6) recycling