Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis.

Stefano Donini,Keigo Shibayama,Davide M Ferraris,Menico Rizzi,Shigetarou Mori,Riccardo Miggiano,Silvia Garavaglia,Eugene A Permyakov

doi:10.1371/journal.pone.0175815

Abstract

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

Highlights

M. tuberculosis (MTB), the causative agent of tuberculosis (TB), is a pathogen as old as the human species that, even today, continues to be a threat for the entire world population
Compared to its ortholog in M. tuberculosis [8,14], the recombinant MsPrsA did not show any aggregate or any different oligomerization form than an hexameric quaternary structure typical of the Class I phosphoribosylpyrophosphate synthase [31,32], as shown by the lack of peaks corresponding to the column exclusion volume, and by the eluition volume of the peak corresponding to the eluted protein (Fig 1)
We expected MsPrsA to belong to the Class I phosphoribosylpyrophosphate synthase subfamily, as previously demonstrated for the strict ortholog M. tuberculosis enzyme [8,13].as expected for a Class I phosphoribosylpyrophosphate synthetase (PrsA) [33,34], we show that the M. smegmatis enzyme requires phosphate ion (Pi) with an optimal concentration ranging from 30 mM to 50 mM, with maximal activity peaking at 50 mM (Fig 2A)

Summary

Introduction

M. tuberculosis (MTB), the causative agent of tuberculosis (TB), is a pathogen as old as the human species that, even today, continues to be a threat for the entire world population. An additional complication for the treatment of TB is due to the ability of MTB to thrive in a dormant state in apparently healthy individuals, causing the asymptomatic infected carrier to be unaware of the disease, while retaining instead a pool of dormant bacteria in the body that can possibly resuscitate and that represent the reservoir of the disease. Such a dormant condition is overturned whenever an immunosuppressive condition occurs, as .

Methods

Results

Conclusion