Abstract
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.
Highlights
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)
PNPOx is able to use both PNP and pyridoxamine 5Ј-phosphate (PMP) as substrates; the E. coli enzyme, contrary to its mammalian counterpart that shows similar catalytic efficiency with both substrates, oxidizes PNP with a catalytic efficiency that, at pH 7.6 (which is the internal pH of E. coli cells [18]), is about 50-fold higher [13]. This is consistent with the peculiar role played by PNPOx in E. coli, where PNP oxidation is the last step of PLP de novo biosynthesis
When we carried out PNPOx activity measurements in a different buffer (50 mM NaHEPES, pH 7.6), in which free PLP is allowed to accumulate in the solvent, we observed complex kinetics that could not be accounted for by simple competitive product inhibition
Summary
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). PLP product inhibition of E. coli PNPOx has been reported to take place with a KI of 8 M [13] and attributed to PLP binding at the active site; data supporting the competitive nature of product inhibition with respect to the PNP substrate have not been presented This product inhibition is probably an important regulatory mechanism of PLP biosynthesis in E. coli cells, whose free PLP concentration in vivo has been estimated around 120 M [14]. We present data showing that, in net contrast with previous reports, inhibition of E. coli PNPOx by PLP does not result from binding of this vitamer at the active site but from binding at an allosteric site
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