Pyridoxal-5-phosphate (PLP) is a highly conserved cofactor used by thousands of known enzymes to carry out biological reactions. Because of its high conservation, the synthesis of PLP has been extensively studied over the years. The PLP synthase complex is an enzyme composed of two catalytic domains, Pdx1 and Pdx2, that work together in the biosynthesis of PLP. Pdx2 catalyzes glutamine hydrolysis to free ammonia, which Pdx1 uses in addition to glyceraldehyde-3-phosphate (G3P) and Ribose-5-phosphate (R5P) to arrive at the final product (figure 1a). The Pdx1 domain contains two phosphate binding sites, P1 and P2, that are used to hold on to the R5P phosphate when the reaction is initiated, and the PLP phosphate at the conclusion of the reaction. Knowing that P1 is the substrate binding site and P2 is the product binding site, and that the two sites are 21 Å apart (figure 1b) suggests the presence of a mechanism in which Pdx1 transfers the intermediate from one site to the other without disrupting the reaction. In their recent Nature paper, Rodriguez et all. propose a mechanism by which this complex reaction is shuttled, within a single domain, with the help of two conserved Lysine residues. Findings in this paper not only add to the field’s knowledge of substrate-channeling mechanisms, but are also the first to show how Lysine imines participate in such mechanisms.
Intricate biochemical reactions such as the oxidative decarboxylation of pyruvate or the biosynthesis of PLP, are known to be catalyzed by enzyme complexes. An enzyme complex may be composed of multiple enzymes or one enzyme with multiple associated polypeptide chains, depending on the organism. In eukaryotes, the latter is more prevalent. In contrast, prokaryotes use multiple enzymes to carry out similar reactions. In eukaryotes, private dehydrogenase and other similar enzyme complexes use cofactors, lipoaminde in this case, to transfer reaction intermediates from one domain (E1) to another (E2) as shown in figure 2. The authors of the paper, however, point out that PLP synthase is different from previously studied enzyme complexes in that it transfers intermediates within a single catalytic domain.
To study the role of Lysine in the shuttling of the intermediates during PLP biosynthesis, the authors conducted crystallographic studies of the Arabidopsis thaliana Pdx1. In order to obtain accurate structures of enzyme-substrate complexes, the authors performed soaking experiments to more than 1,000 crystals, which resulted in the identification of five covalent intermediates: Pdx1-R5P, K166R-pre-I320, Pdx1-I320, Pdx1-I320-G3p, and Pdx1-PLP (figure 3).
Biosynthesis of PLP begins when the R5P phosphate binds to Lys98 in P1, forming the first intermediate Pdx1-R5P. Ammonia reacts with Pdx1-R5P to form what the authors call a “chromophoric I320” intermediate. The formation of this intermediate causes conformational changes in Pdx1 that result in the diffusion of R5P out of the P1 site. With the P1 site empty, G3P is allowed to bind to form Pdx1-I320-G3P. Note the dual-specificity nature of the P1 binding site. The structure of the Pdx1-I320-G3P intermediate is what was most shocking to the authors, as I320 was covalently bound to both Lys98 and Lys166. The final structure of the product was identified When Pdx1 crystals were soaked with PLP. Pdx1-PLP was covalently attached to Lys166 only.
Due to the conformational changes that are necessary for the reaction, the authors ran into many problems associated with the crystal soaking technique. When crystals are formed, structural rearrangement are halted, which can result in less inaccurate data when used in experiments like the one conducted by Rodrigues et al. Perhaps in further studies, techniques like co-expression of the protein with the ligands of interest, use of the ligands during protein purification, and co-crystallization can be considered to strengthen the author’s arguments.
Intermediate transfer strategies allow for more efficient catalysis of reactions that would otherwise require several enzymes. Transimination, as many of us have learned, is a crucial PLP dependent reaction that allows for amino acid metabolism, but it is only one of the hundreds of reactions that require PLP. Studying the biosynthesis of such a highly conserved cofactor will eventually lead to the discovery of novel targets for drug therapy for illnesses such as Pyridoxamine-5-phosphate oxidase deficiency which leads to low PLP concentrations in those affected.
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