title: Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.
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.
authors: Scientific Institute of Public Health, Direction of Communicable and Infectious Diseases, Rue Engeland 642, 1180 Brussels, Belgium.
authors: Xiao Ming
authors: XM
authors: Wang
authors: Scientific Institute of Public Health, Direction of Communicable and Infectious Diseases, Rue Engeland 642, 1180 Brussels, Belgium.
authors: Karine
authors: K
authors: Soetaert
authors: Scientific Institute of Public Health, Direction of Communicable and Infectious Diseases, Rue Engeland 642, 1180 Brussels, Belgium.
authors: Priska
authors: P
authors: Peirs
authors: Scientific Institute of Public Health, Direction of Communicable and Infectious Diseases, Rue Engeland 642, 1180 Brussels, Belgium.
authors: Michaël
authors: M
authors: Kalai
authors: Unité de Microbiologie Pharmaceutique et Hygiène, Faculty of Pharmacy, Université Libre de Bruxelles, Boulevard du Triomphe, CP205/2, 1050 Brussels, Belgium.
authors: Véronique
authors: V
authors: Fontaine
authors: Unité de Microbiologie Pharmaceutique et Hygiène, Faculty of Pharmacy, Université Libre de Bruxelles, Boulevard du Triomphe, CP205/2, 1050 Brussels, Belgium.
authors: Jean Paul
authors: JP
authors: Dehaye
authors: Unité de Microbiologie Pharmaceutique et Hygiène, Faculty of Pharmacy, Université Libre de Bruxelles, Boulevard du Triomphe, CP205/2, 1050 Brussels, Belgium.
authors: Philippe
authors: P
authors: Lefèvre
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D008291:Malate Dehydrogenase; D011494:Protein Kinases; C112455:PknD protein, Nostoc sp. PCC 7120; D017346:Protein Serine-Threonine Kinases