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Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase, a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis.

Authors :
Wang, Xiao Ming
Soetaert, Karine
Peirs, Priska
Kalai, Michaël
Fontaine, Véronique
Dehaye, Jean Paul
Lefèvre, Philippe
Source :
PLoS ONE; Apr2015, Vol. 10 Issue 4, p1-17, 17p
Publication Year :
2015

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<superscript>+</superscript>-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. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
19326203
Volume :
10
Issue :
4
Database :
Complementary Index
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
102402074
Full Text :
https://doi.org/10.1371/journal.pone.0123327