Abstract
Vitamin B(6) is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5'-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A glutamine amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and glutamine.
Highlights
We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx[1], and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx[2] and glutamine
It was long assumed that de novo biosynthesis of vitamin B6 could only be achieved by the DXP4-dependent pathway, characterized extensively for Escherichia coli (1, 2), which generates the active cofactor form via pyridoxine 5Ј-phosphate from erythrose 4-phosphate and glyceraldehyde 3-phosphate through a sequence of reactions requiring the participation of seven enzymes
Employing fluorescence and UV-visible spectrometry combined with protein mutagenesis, we demonstrate that the C terminus of Pdx[1] is essential in mediating these functionalities in pyridoxal 5Ј-phosphate (PLP) synthase, and we provide insight into the dynamics of this highly flexible region, which has a major role to play in the mechanism of this polymorphic enzyme
Summary
Materials—p-Benzoyl-phenylalanine (pBpa) was purchased from Bachem AG (Bubendorf, Switzerland). Nano-liquid Chromatography Separation and MALDI Analysis of Tryptic Peptides—One hundred M Pdx[1] protein sample (wild type or W294:pBpa in a total volume of 100 l that had been subjected to 60 min of irradiation at 365 nm using the UV Stratalinker 1800) was digested with 2 g of trypsin for 3 h at 37 °C in 100 mM NH4HCO3. The mutant Pdx1 ⌬W294 was generated using the QuikChange site-directed mutagenesis kit (Stratagene, Basel, Switzerland) employing the oligonucleotides 5Ј-GCAAGAACGCGGCCTCGAGCACCACC-3Ј and 5Ј-GGTGGTGCTCGAGGCCGCGTTCTTGC-3Ј as forward and reverse primer, respectively, and pETBsPdx1-His[6] as the template. In order to relate these measurements with the observed changes in intrinsic tryptophan fluorescence, identical measurements were carried out following tryptophan fluorescence essentially as described above with the exception that 1 M Pdx[1] and Pdx[2] H170N were used
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