Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.

Philippe Lefèvre,Xiao Ming Wang,Priska Peirs,Véronique Fontaine,Karine Soetaert,Michaël Kalai,Jean Paul Dehaye,Joyoti Basu

doi:10.1371/journal.pone.0123327

Philippe Lefèvre, Xiao Ming Wang + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0123327

Copy DOI

Abstract

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.

Highlights

Mycobacterium tuberculosis (Mtb), a causative agent of tuberculosis, is responsible for considerable worldwide human morbidity and mortality
These results suggest that the malate dehydrogenase (MDH) is a substrate of several mycobacterial serine/threonine protein kinases (STPKs), the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions
To investigate the substrates of the PknD protein kinase, a soluble extract of M. bovis BCG was applied to a phenyl-Sepharose column CL-4B and the unbound pass-through material was separated by anion-exchange chromatography on a Resource-Q column

Summary

Introduction

Mycobacterium tuberculosis (Mtb), a causative agent of tuberculosis, is responsible for considerable worldwide human morbidity and mortality. A third of the world population is infected with persistent or latent Mtb, referred to as latent tuberculosis. Reactivation of latent infection is the major source of active tuberculosis in adults [1]. One of the main obstacles in the global control of tuberculosis is linked to the emergence of multi-drug resistant strains and the therapeutic failure of persistent infections treatment using conventional anti-tuberculosis. Mtb PknD Phosphorylates the Malate Dehydrogenase drugs. Mtb resides in the hypoxic necrotic core of complex immunological structures, called granulomas, either within macrophages or in the extracellular compartment [2]

Methods

Results

Conclusion