An Anaerobic-Type α-Ketoglutarate Ferredoxin Oxidoreductase Completes the Oxidative Tricarboxylic Acid Cycle of Mycobacterium tuberculosis

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Abstract
Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable α-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO2. Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of β-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with β-oxidation (KOR-dependent), and one that functions in the absence of β-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host. Knowledge of the basic biology of Mycobacterium tuberculosis is essential to identifying novel ways to combat the emerging threat of drug-resistant tuberculosis. Since the tricarboxylic acid (TCA) cycle is a cornerstone of metabolism and M. tuberculosis does not possess a “typical” TCA cycle enzyme set, much effort has been focused on elucidating the components of this pathway. Previous reports indicate that M. tuberculosis possesses a variant TCA cycle in which succinic semialdehyde replaces succinyl-CoA. Since this pathway does not conserve as much metabolic energy as the canonical pathway, we considered an alternative hypothesis: that M. tuberculosis might possess an anaerobic type α-ketoglutarate dehydrogenase. In this manuscript, we investigate this previously unknown activity for mycobacteria using a combination of genetic and biochemical approaches, and demonstrate that M. tuberculosis is capable of driving a conventional TCA cycle in an unconventional way. We also validate the existence of the previously described variant pathway and provide evidence that these two pathways are differentially utilized in response to a metabolic signal, fatty acid catabolism.

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