Figure 1.
PLP formation catalyzed by PNPOx and associated vitamin B6 metabolic pathways.
The role of Rv2607 is shown in red. In E. coli, PNPOx catalyzes the last step in the DXP-dependent PLP biosynthetic pathway [2]. Most organisms capable of PLP biosynthesis produce PLP via PLP synthase, a macromolecular complex consisting of Pdx1 and Pdx2 [2]. Organisms with genes that encode both PLP synthase and PNPOx likely use PNPOx to salavage PLP from PNP and PMP, which are produced by enzymes that use PLP as a cofactor.
Table 1.
Summary of NCBI gene annotations for PNPOx-like proteins in selected organisms that contain pdx1 and pdx2 and the role of PNPOx is considered to be for PLP salvage.
Figure 2.
Nano-ESI mass spectrum of intact Rv2607 with co-purified FMN.
The major species, C, has peaks with charges ranging from 15+ to 12+ in the 3500-4500 m/z range that correspond to a molecular weight of 55376±12. This experimental mass is consistent with the calculated mass for the complex of dimeric Rv2607 with one molecule of FMN bound (55442 Da), which is derived from the amino acid sequence of the 6x-histidine tagged Rv2607 (54986 Da; 27493 Da per protomer) and the molecular weight of FMN (456 Da). Monomeric tagged Rv2607 and truncated Rv2607 monomer (missing 10-11 residues), are present in solution and correspond to molecular weights of A (27450±52 Da) and B (26215±54 Da) respectively. A minor dimeric species comprised of full length and truncated monomer is represented as species D. Higher molecular weight oligomers (trimers E and tetramers F with molecular weights of 110.6 and 82.7 kDa, respectively) are electrospray-induced non-specific association of monomers and dimers.
Figure 3.
Reverse-phase HPLC, NMR, and spectrophotometric analysis of the PNPOx activity of Rv2607.
(A) HPLC chromatograms (270 nm) of reaction mixtures containing PNP (i-iv) or PMP (v-viii), FMN, and Rv2607, and the associated control reactions. All reactions were carried out in 25 mM potassium phosphate buffer (pH 7.8), incubated at 25°C for 3 h, and quenched with DNPH (0.7 mM final concentration). Where present, the reaction components were at the following concentrations: 1 mM PNP or PMP, 10 µM FMN, and 10 µM Rv2607. Reaction mixtures contained: (i) PNP, FMN, and Rv2607, (ii) PNP and FMN (enzyme negative control), (iii) PNP and Rv2607 (no added FMN), (iv) FMN and Rv2607 (substrate negative control), (v) PMP, FMN, and Rv2607, (vi) PMP and FMN (enzyme negative control), (vii) PMP and Rv2607 (no added FMN), (viii) FMN and Rv2607 (substrate negative control). The peak to the right of PLP-DNPH is DNPH, which has a retention time of 8.6 min. (B) 1H NMR analysis of the conversion of PNP into PLP with time. The enzymatic reaction (1 mM PNP, 28 µM enzyme, 10% D2O in 25 mM potassium phosphate buffer, pH 7.8) was incubated for 18 hours at 298 K in the spectrometer. Left; stacked 1H NMR spectra recorded at various time points and after the addition of authentic PLP. The starred peak corresponds to PLP hydrate. Right; a plot of percent substrate conversion versus time. Substrate conversion was determined by comparing integrals of the C2-1H signals associated with PLP (7.80 ppm) to that of PNP (7.75 ppm). (C) Michaelis-Menten plot for Rv2607 with PNP as a substrate. The rate of PLP formation was monitored spectrophotometrically (λmax = 388 nm, ε = 4900 cm−1M−1) for various concentrations of PNP. All solutions were made in 100 mM potassium phosphate buffer (pH 7.8).