Functional importance of Asp264 in the ketosynthase domain of Pks13 in Mycobacterium smegmatis.

Mycolic acids are key components of the mycobacterial cell envelope, contributing to its structural integrity and intrinsic resistance to environmental stress. The polyketide synthase Pks13 catalyzes the final Claisen condensation step in mycolic acid biosynthesis and is conserved across Corynebacteriales. While the catalytic cysteine within the ketosynthase (KS) domain is established as essential for activity, the role of surrounding residues in supporting catalysis remains less well understood. Herein, we utilized a Mycobacterium smegmatis conditional pks13 mutant, and subsequent complemented strains harbouring a plasmid-borne copy of pks13 to probe the functional importance of selected residues within the KS domain from previous structural studies. These site-directed mutagenesis studies identified Asp264 as critical for Pks13 function. The D264A strain showed severely impaired growth along with a pronounced loss of mycolate synthesis and mycolate-containing lipids: trehalose dimycolate (TDM) and trehalose monomycolate (TMM). In contrast, other mutations had either little or no significant effect on cell viability or mycolate synthesis or mycolate-containing lipids. Structural modelling of the KS domain suggests that loss of Asp264 disrupts the local active-site environment, resulting in compaction of the binding pocket and altered conformation of a loop proximal to the catalytic residue Cys267. Together, these findings demonstrate that Asp264 is essential for Pks13 function and likely contributes to maintaining the structural environment of the KS domain.