Diacyltransferase Activity and Chain Length Specificity
of <i>Mycobacterium tuberculosis</i> PapA5 in the Synthesis
of Alkyl β‑Diol Lipids
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Abstract
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