Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl β-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl β-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.