Elucidating biosynthesis of the outer membrane lipid phthiocerol dimycocerosate by Mycobacterium tuberculosis PapA5 (768.13)

Abstract

Mycobacterium tuberculosis (Mtb) has a complex, asymmetric outer membrane consisting of mycolic acids and other unique lipids. Phthiocerol dimycocerosate (PDIM) is a wax ester lipid found in the outer membrane, at the interface between the bacterium and host, where it has been implicated in Mtb virulence. My work focuses on gaining a better understanding of how PDIM biosynthesis is regulated in Mtb by studying PapA5, an acyltransferase responsible for acylating phthiocerol with mycocerosic acid. We show using a series of synthetic substrates that PapA5 has a preference for longer alkyl chains. We demonstrate under novel biphasic reaction conditions that PapA5 is capable of catalyzing two successive acylation reactions, consistent with its proposed role in modifying both hydroxyls on the phthiocerol moiety. The introduction of bulky amino acid side chains in the putative substrate‐binding pocket of PapA5 yielded partial or complete inhibition of product formation. These data help confirm the location of the substrate‐binding channels. Furthermore, the introduction of a salt bridge or a disulfide bond to constrain a hypothesized flexible helix led to partial inhibition of product. These data support a role for conformational flexibility in ligand binding and/or product release. Finally, in vitro phosphorylation of PapA5 altered product yield, demonstrating a possible mechanism of regulation for PDIM biosynthesis.Grant Funding Source: Supported by a Stony Wold‐Herbert Fund award to JCS