Determination of Phosphorylation Sites for NADP-specific Isocitrate Dehydrogenase from Mycobacterium tuberculosis

Abstract

Isocitrate Dehydrogenase (ICD) catalyzes the oxidative decarboxylation reaction of 2R,3S- isocitrate to yield 2-oxoglutarate in the Tricarboxylic Acid (TCA) cycle. Two isoforms of NADP-specific ICDs with the E.C number 1.1.1.42 have been annotated in the organism Mycobacterium tuberculosis, monomeric ICD2 and dimeric ICD1. BLAST search against the Protein Data Bank (PDB) database shows a marked similarity between dimeric Mycobacterium tuberculosis ICD1 sequence and that of Sus scrofa, a cytosolic eukaryotic ICD (65% identity). Escherischia coli ICD shows less sequence similarity than the eukaryotic structure. A Homology model has thus been built for M. tuberculosis ICD1 using Sus scrofa and human ICD as templates. Inactivation of ICD1 by phosphorylation similar to E. coli ICD is important to open up the shunt pathway in the TCA cycle, which has been indicated in the case of M. tuberculosis. We therefore attempted to identify a number of likely phosphorylation sites in M. tuberculosis using pattern prediction and checked with the homology models for the accessibility of the peptides containing Serine. It was found that the homologous Serine by alignment with E. coli on M. tuberculosis ICD1 is difficult to access by specific kinases. Hence other probable sites of phosphorylation were checked and three highly probable serine-containing peptides were identified. The effect of phosphorylation at each of these sites was determined by checking the degree of conformational changes, the differences caused by the effect of phosphorylation in the active-site and other apparent motion different from that of the control, i.e., unphosphorylated M. tuberculosis ICD1 model, using molecular dynamics simulations.