Variant tricarboxylic acid cycle in Mycobacterium tuberculosis : Identification of α-ketoglutarate decarboxylase

Abstract

Mycobacterium tuberculosis (Mtb) has adapted its metabolism for persistence in the human macrophage. The adaptations are likely to involve Mtb's core intermediary metabolism, whose enzymes have been little studied. The tricarboxylic acid cycle is expected to yield precursors for energy, lipids, amino acids, and heme. The genome sequence of Mtb H37Rv predicts the presence of a complete tricarboxylic acid cycle, but we recently found that ␣-ketoglutarate dehydrogenase (KDH) activity is lacking in Mtb lysates. Here we showed that citrate synthase, aconitase, isocitrate dehydrogenase, fumarase, malate dehydrogenase, and succinate dehydrogenase, but not KDH, are present, raising the possibility of separate oxidative and reductive half-cycles. As a potential link between the half-cycles, we found that Rv1248c, annotated as encoding SucA, the putative E1 component of KDH, instead encodes ␣-ketoglutarate decarboxylase (Kgd) and produces succinic semialdehyde. Succinic semialdehyde dehydrogenase activity was detected in Mtb lysates and recapitulated with recombinant

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

“…Rv1248c by itself produces succinate semialdehyde (SSA) from nonoxidative decarboxylation of α-KG in vitro, and Mtb's succinate semialdehyde dehydrogenases (SSADHs) can generate succinate from SSA, completing a modified TCA cycle (13). Subsequently, activity-based metabolomic profiling revealed yet another function of Rv1248c that predominated over SSA production: decarboxylation of α-KG, followed by carboligation with glyoxylate to form 2-hydroxy-3-oxoadipate (HOA).…”

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…In view of results so far accumulated (6,7,26), it appears that pathways 1 and 2 are predominant. All three pathways are derived from ThDP-catalyzed decarboxylation of 2-ketoglutarate and the reaction of the enamine intermediate with either a H ϩ , glyoxylate, or lipoamide bound to a putative E2, respectively.…”

“…Plate Reader Assays-A typical ferricyanide reductase assay reaction mixture (200 l) in 20 mM Bis-Tris (pH 6.5) contained 5 mM MgCl 2 , 1.6 mM K 3 Fe(CN) 6 …”

“…Ferricyanide reductase assays were performed as described (6). Succinate semialdehyde production was monitored using horse liver alcohol dehydrogenase in the presence of NADH, and the disappearance of NADH was monitored at 340 nm.…”

“…In mycobacterial cell extracts, the 2-ketoglutarate dehydrogenase complex activity could not be detected, and the E2o component could not be identified (6). However, the product of the gene Rv1248c (annotated as the E1o of Mycobacterium tuberculosis 2-ketoglutarate dehydrogenase complex) could catalyze the synthesis of HOA in vitro using either 2-ketoglutarate and glyoxylate or a small molecule extract of mycobacteria as a source of substrates.…”