Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we employed genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium. The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates export of MAs and promotes remodeling of the mycobacterial cell envelope. In particular, we identified the essential mycobacterial membrane protein large 3 (MmpL3) as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way towards novel antimycobacterial strategies.
Tuberculosis (TB), caused by
Mycobacterium tuberculosis
, is one of the leading causes of death worldwide. The rise in drug resistance highlights the urgent need for innovation in anti-TB drug development.
The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacyl catalysis intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site allowed us to boost the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.
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