Dissecting the Ca²⁺ dependence of DesA1 function in Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca²⁺-binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca²⁺ binding, we introduced mutations at key residues in its Ca²⁺-binding βγ-crystallin motif to generate DesA1F303A, E304Q, and F303A-E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca²⁺ non-binders F303A or F303A-E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca²⁺ in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity.