O2- and NO-sensing mechanism through the DevSR two-component system in Mycobacterium smegmatis

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• The DevS protein in Mycobacterium smegmatis is a histidine kinase with N-terminal tandem GAF domains.
• DevS possesses a heme iron in its ferrous state, which is resistant to autooxidation.
• Sequence analysis suggests DevS is more closely related to Mycobacterium tuberculosis DosT.

Purpose of the Study:

• To investigate the role of the heme iron in DevS autokinase activity regulation.
• To determine the effect of oxygen (O2) and nitric oxide (NO) binding on DevS activity.
• To explore potential regulatory mechanisms involving cyclic nucleotides and the respiratory chain.

Main Methods:

• Purification of DevS and assessment of its heme iron redox state.
• Analysis of DevS autokinase activity upon O2 and NO binding.
• Characterization of DevS derivatives lacking heme.
• Structural analysis of the GAF-B domain.
• Investigation of cyclic nucleotide and respiratory chain involvement.

Main Results:

• Oxygen binding to deoxyferrous heme decreased DevS autokinase activity; NO binding had no effect.
• DevS derivatives lacking heme showed no O2 or NO-dependent regulation, highlighting the heme's importance.
• The quinone/quinol pool and cyclic nucleotides (cGMP, cAMP) did not regulate DevS activity.
• The GAF-B domain exhibits a GAF fold but lacks cyclic nucleotide binding capacity.

Conclusions:

• The heme iron's ligand-binding state is critical for regulating DevS autokinase activity in response to O2.
• DevS regulation is distinct from mechanisms involving cyclic nucleotides or the respiratory electron transport chain.
• DevS represents a unique heme-based sensor kinase in Mycobacterium species.