Structural enzymological studies of the long chain fatty acyl-CoA synthetase FadD5 from the mce1 operon of Mycobacterium tuberculosis.

The cell wall of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is rich in complex lipids. During intracellular stage, Mtb depends on lipids for its survival. Mammalian cell entry (Mce) 1 complex encoded by the mce1 operon is a mycolic/fatty acid importer. mce1 operon also encodes a putative fatty acyl-CoA synthetase (FadD5; Rv0166), potentially responsible for the activation of fatty acids imported through the Mce1 complex by conjugating them to Coenzyme A. Here, we report that FadD5 is associated to membrane although it can be purified as a soluble dimeric protein. ATP and CoA binding influence FadD5's stability and conformation respectively. Enzymatic studies with fatty acids of varying chain lengths show that FadD5 prefers long chain fatty acids as substrates. X-ray crystallographic studies on FadD5 and its variant reveal that the C-terminal domain (∼100 residues) is cleaved off during crystallization. Noteworthy, deletion of this domain renders FadD5 completely inactive. SAXS studies, however, confirm the presence of full length FadD5 as a dimer in solution. Further structural analysis and comparisons with homologs provide insights on the possible mode of membrane association and fatty acyl tail binding.