title: Targeting lipid esterases in mycobacteria grown under different physiological conditions using activity-based profiling with tetrahydrolipstatin (THL).
abstract: Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456;
authors.forename: Madhu Sudhan
authors.initials: MS
authors.lastname: Ravindran
authors.forename: Srinivasa P S
authors.initials: SP
authors.lastname: Rao
authors.forename: Xiamin
authors.initials: X
authors.lastname: Cheng
authors.forename: Ankit
authors.initials: A
authors.lastname: Shukla
authors.forename: Amaury
authors.initials: A
authors.lastname: Cazenave-Gassiot
authors.forename: Shao Q
authors.initials: SQ
authors.lastname: Yao
authors.forename: Markus R
authors.initials: MR
authors.lastname: Wenk
mesh_terms: D001431:Bacteriological Techniques; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004950:Esterases / Q000037:antagonists & inhibitors*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007783:Lactones / Q000494:pharmacology*; D008049:Lipase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050356:Lipid Metabolism / Q000187:drug effects; D058990:Molecular Targeted Therapy; D009163:Mycobacterium bovis / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development* / Q000378:metabolism; D000077403:Orlistat; D014280:Triglycerides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007783:Lactones; D014280:Triglycerides; D000077403:Orlistat; D004950:Esterases; D008049:Lipase