Dissecting the Ca²⁺dependence ofMycobacterium tuberculosisDesA1 function

Mycobacterium tuberculosis(M. tb) has a complex cell wall, largely composed of mycolic acids and long-chain fatty acids that play a crucial role in maintaining its integrity and permeability. This complex lipid structure has a role in abrogating the process of phagosome-lysosome fusion and infection establishment. TheM. tbdesaturase A1 (DesA1) catalyzes the introduction of position-specific double bonds, a key step in the biosynthesis of a diverse range of mycolic acids. We have previously demonstrated thatM. tbDesA1 is a Ca2+-binding protein, belonging to the extended βγ-crystallin superfamily. Using a combination of biophysical and genetic approaches, we investigated the structural and functional significance of Ca2+binding on DesA1 activity. A protein unfolding assay of the protein in the presence and absence of Ca2+shows that Ca2+binding imparts structural stability to DesA1. To identify the role of Ca2+, we introduced mutations at key residues in the identified Ca2+-binding motif of DesA1 and generated F303A, E304Q, and F303A-E304Q variants of DesA1. We identified F303 as a hot point which disables the protein for Ca2+binding. Two other mutations E304Q and F303A-E304Q showed reduced Ca2+binding. Complementation of a conditionally complementeddesA1deletion mutant strain ofMycobacterium smegmatiswith these mutants, either failed to complement its growth phenotype or led to a compromise in complementation. In addition, the F303A and F303A-E304Q complements exhibit increased sensitivity to isoniazid, a first-line anti-tubercular drug, pointing to a cell wall permeability defect in these strains. Our findings highlight the critical importance of Ca2+in the functioning of DesA1 and its implicit role in the maintenance of mycobacterial cellular integrity.