Solution Structures and Dynamic Assembly of the 24-Meric Plasmodial Pdx1-Pdx2 Complex.

Najeeb Ullah,Celestin Nzanzu Mudogo,Hina Andaleeb,Carsten Wrenger,Sven Falke,Christian Betzel

doi:10.3390/ijms21175971

Abstract

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host’s metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1–Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.

Highlights

Malaria, with more than 250 million humans infected annually and up to 0.5 million fatalities [1], highlights an urgent need to identify and discover new antimalarial drugs [2] for use against the human pathogens Plasmodium falciparum, P. vivax, Plasmodium knowlesi, Plasmodium malariae, and Plasmodium ovale, in particular [2,3]
For the P. vivax Pdx complex, we showed by applying size-exclusion chromatography (SEC), dynamic light scattering (DLS), small angle X-rays scattering (SAXS), and electron microscopy (EM) that the complex was stable in solution without observing higher-order oligomers over time
We presented a detailed analysis of the P. vivax Pdx proteins, Pdx1 and Pdx2 in solution, that form the pyridoxal phosphate (PLP) biosynthesis enzyme complex and provided structural details about the P. vivax Pdx1 dodecamer, the oligomerization behavior of Pdx2, and the dynamics of Pdx complex formation

Summary

Introduction

With more than 250 million humans infected annually and up to 0.5 million fatalities [1], highlights an urgent need to identify and discover new antimalarial drugs [2] for use against the human pathogens Plasmodium falciparum, P. vivax, Plasmodium knowlesi, Plasmodium malariae, and Plasmodium ovale, in particular [2,3]. Drug targets that interfere with the metabolism of the parasite, like the vital vitamin pathways, are the focus of the latest drug discovery investigations [4]. P. falciparum is responsible for the majority of the cases and deaths, P. vivax has a wider geographical distribution and is the causative organism of almost half of malaria cases outside Africa. It generates re-emerging quiescent liver-stage parasites, causing repeated clinical episodes of malaria due to a single infection. Pyridoxal-5-phosphate (PLP), the active cofactor for more than 100 vitamin B6-dependent enzymes, results from the transformation of its precursors, namely pyridoxine, pyridoxamine, and pyridoxal. The ammonia produced by Pdx is supplied to the active site of the synthase domain (Pdx1) through a hydrophobic ammonia tunnel [12]

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Conclusion