DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis.

Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N-terminal GAF domain. For full-length DosT and DevS, we determined the ligand-binding parameters and the rates of ATP reaction with the liganded and unliganded states. In both proteins, the heme state was coupled to the kinase such that the unliganded, CO-bound, and NO-bound forms were active, but the O(2)-bound form was inactive. Oxygen-bound DosT was unusually inert to oxidation to the ferric state (half life in air >60 h). Though the kinase activity of DosT was unaffected by NO, this ligand bound 5000 times more avidly than O(2) to DosT (K(d) [NO] approximately 5 nM versus K(d) [O(2)] = 26 microM). These results demonstrate direct and specific O(2) sensing by proteins in M. tuberculosis and identify for the first time a signal ligand for a sensory kinase from this organism. They also explain why exposure of M. tuberculosis to NO donors under aerobic conditions can give results identical to hypoxia, i.e., NO saturates DosT, preventing O(2) binding and yielding an active kinase.

A memory of oxygen binding explains the dose response of the heme-based sensor FixL.

Bradyrhizobium japonicum FixL is a modular oxygen sensor that directs adaptations to hypoxia by coupling the status of a heme-binding domain to a histidine-protein kinase activity. The oxygen-bound form is the "off-state". The unliganded form is the "on-state" active kinase that phosphorylates a transcription factor, FixJ. We have developed methods to optimize the kinase reactions of FixL and measure the turnover rates (kcat) for reactions catalyzed by highly inhibited states of this sensor at constant, precisely known oxygen saturations. The resulting oxygen dose-response curve shows that an in vitro system with FixL and the response regulator FixJ as its only proteins manifests such a sharp ligand response that, when the proportion of deoxy-FixL decreases less than 3-fold, the kinase activity drops over 50-fold, and by the time the deoxy-FixL declines just 8-fold, the activity is inhibited over 1100-fold. This response is entirely reversible and similar to that reported for the in vivo hypoxic induction of FixLJ-regulated genes. FixL binds oxygen noncooperatively. When complexed with FixJ, FixL is dimeric in both oxy and deoxy states. Therefore traditional models involving cooperative binding of ligand or robust allosteric regulation cannot account for the extremely nonlinear kinase response to the heme saturation. This response, however, can be explained by a form of enzyme hysteresis with the simple assumptions that (i) on association of oxygen with the heme, the kinase is rapidly switched off; (ii) after dissociation of oxygen, the kinase remains inhibited longer than the average time that it takes a deoxy-heme to encounter an oxygen molecule at most oxygen saturations.