| Mycobacterium tuberculosis (Mtb) persists for prolonged periods in macrophages, where it must adapt to metabolic limitations and oxidative/nitrosative stress. However, little is known about Mtb's intermediary metabolism or anti-oxidant defenses. Our group recently discovered a peroxynitrite reductase-peroxidase complex in Mtb that included products of the genes sucB and lpd, which are annotated to encode the dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3) components of alpha-ketoglutarate dehydrogenase (KDH). We examined whether these proteins identified in the Mtb's antioxidant defense pathways actually play a role in Mtb's core intermediary metabolism.; However, we could detect no KDH activity in Mtb lysates, nor could we reconstitute KDH by combining the recombinant proteins SucA (annotated as the E1 component of KDH), SucB and Lpd. Mtb lysates contained pyruvate dehydrogenase (PDH) activity, which was lost when the dlaT gene (formerly, sucB) was disrupted, thus indicating that SucB is part of PDH. We therefore renamed the sucB product dihydrolipoamide acyltransferase (DlaT). To identify other components of PDH, we partially purified PDH from Mtb and by mass peptide finger printing identified AceE, annotated as an E1 component of PDH, along with DlaT and Lpd. Moreover, anti-DlaT antibody coimmunoprecipitated AceE. Finally, recombinant AceE, DlaT and Lpd, although encoded by genes that are widely separated on the chromosome, reconstituted PDH in vitro with Km values typical of bacterial PDH complexes. In contrast, no PDH activity was generated when Lpd was combined with PdhA, PdhB and PdhC that form a gene cluster and are annotated as encoding PDH. Hence, our results suggest that Mtb appears to lack KDH and encodes PDH with widely separated genes, aceE, dlaT and lpd.; Lack of KDH, although reported for other bacteria, has not been demonstrated in Mtb. This directed our attention to Mtb's tricarboxylic acid (TCA) cycle. TCA cycle is expected to yield precursors for energy, lipids, amino acids and heme. But Mtb's TCA cycle enzymes have been little studied. The genome sequence of Mtb H37Rv predicts the presence of a complete TCA cycle, but we detect no KDH in Mtb lysates. We surveyed the remaining TCA cycle enzymes and sought additional enzymes that might bridge the cycle in the absence of KDH. Citrate synthase, aconitase, isocitrate dehydrogenase, fumarase, malate dehydrogenase and succinate dehydrogenase activities were detected, but no KDH was present. This suggests that Mtb might possess separate oxidative and reductive half-cycles. (Abstract shortened by UMI.); Part of the abstract was taken from Jing Tian, Ruslana Bryk, Shuangping Shi, Hediye Erdjument-Bromage, Paul Tempst and Carl Nathan. Molecular Microbiology (2005) 57(3), 859-868; from Jing Tian, Ruslana Bryk, Manabu Itoh, Makoto Suematsu and Carl Nathan. PNAS (2005) 102(30), 10670-10675. |
