Assembly and Activation of the Plasmodial Pyridoxal 5’-Phosphate Synthase Complex. Understanding the Structural Mechanism of PLP Biosynthesis

Abstract

Biosynthesis of vitamin B6 is essential for all living cells. Most organisms use the pyridoxal 5’-phosphate (PLP) synthase complex to synthesize the cofactor form, PLP, from the three substrates ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P) and ammonia. PLP synthase complex is a glutamine amidotransferase (GATase) class I, consisting of 12 Pdx1 and 12 Pdx2 subunits. Pdx1 is responsible for the PLP synthesis and Pdx2 is the glutaminase that hydrolyses glutamine to produce ammonia, which is transfered to the Pdx1 active site. In this PhD Thesis, studying Pdx1 and Pdx2 proteins from the human parasite Plasmodium falciparum and from the rodent parasite Plasmodium berghei gave important insights into the assembly, activation and substrate tunneling of the PLP synthase complex. Electron microscopy analyses showed that association of the PLP synthase and glutaminase subunits was random, suggesting a non cooperative mechanism independent of neighboring Pdx1 binding sites to be occupied by Pdx2. Complex assembly is critical for glutamine hydrolysis by Pdx2, although the presence of an ammonia acceptor in the Pdx1 active site did neither enhance the Pdx1/Pdx2 interaction nor the catalytic rate of Pdx2 in vitro, as tested by biophysical and kinetic experiments. In particular, the PLP synthase complex does not show allosteric activation by R5P or glutamine binding that would result in synchronization of the glutaminase and PLP synthesis reactions. Therefore, the Pdx1/Pdx2 interaction is enough to stimulate glutamine hydrolysis. A particular motivation of this Thesis was to crystallize the PLP synthase complex from the malaria causing parasite, P. falciparum, for the potential use of the 3D structure in drug design. However, the complex assembled into fibers in vitro, induced by the Pdx1 subunit, making the crystallization trials of this enzyme impossible. A chimeric complex formed by Pdx1 from P. berghei and Pdx2 from P. falciparum proved to be a catalytically active system, suitable for structural studies of the plasmodial complex. Crystal structure of this enzyme complex gave two major advances in the understanding how prokaryotic and eukaryotic PLP synthase complexes resemble each other or differ in protein interaction and activation. Variations at the Pdx1/Pdx2 interface occur through insertion sequences in eukaryotic systems, notably in the plasmodial PLP synthase complex by the loop 95-111 in Pdx2. Activation of the glutaminase is highly conserved in both systems. The process entails reorganization of structural regions at the Pdx1/Pdx2 interface through stabilization of alpha-N and the oxyanion hole region. Activation of the PLP synthase requires a helical segment, named alpha-2’, for sugar binding. The helix is observed in two alternative positions in the Pdx1/Pdx2 and Pdx1-R5P structures: an open conformation to allow the entrance of the substrate and a closed conformation oriented towards R5P, sequestering the substrate in the catalytic center. The pentose substrate is bound in the P1 site to the catalytic Lys84 via a Schiff base with the ribose C1 atom. GATases are characterized by two separate active sites for glutamine hydrolysis and enzyme-specific metabolite syntheses. Previously, ammonia transfer between two catalytic centers was proposed to occur by flexible methionine residues within a transient tunnel in Pdx1. The plasmodial proteins show an ammonia tunnel distict from bacterial orthologs as some of the residues lining the passage are exchanged. Biochemical analysis confirmed that the (beta/alpha)8 -barrel of Pdx1 passes the reactive ammonia produced in Pdx2 to Pdx1 active site, assigning function of key residues for the ammonia channeling. The differences between eukaryotic and prokaryotic systems provide insight into PLP synthase complex regulation, which may be exploitable in drug design for the treatment of malaria.