Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase.
Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP). This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli and as a part of the salvage pathway of this coenzyme in both E. coli and mammalian cells. Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly. The crystal structures of PNPOx with one and two molecules of PLP bound have been determined. In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN). A large protein conformational change occurs upon binding of PLP. When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site. A possible tunnel exists between the two sites. Site mutants were made of all residues at the active site that make interactions with the substrate. Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP. The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.
di Salvo
Martino L
ML
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, P.le Aldo Moro 5, 00185, via degli Apuli 9, Rome, Italy. martino.disalvo@uniroma1.it
Safo
Martin K
MK
Musayev
Faik N
FN
Bossa
Francesco
F
Schirch
Verne
V
D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D011734:Pyridoxaminephosphate Oxidase / Q000737:chemistry* / Q000378:metabolism
D029968:Escherichia coli Proteins; D011732:Pyridoxal Phosphate; D011734:Pyridoxaminephosphate Oxidase