title: Tetrahydrolipstatin inhibition, functional analyses, and three-dimensional structure of a lipase essential for mycobacterial viability.
abstract: The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia. paul.crellin@monash.edu
authors.forename: Paul K
authors.initials: PK
authors.lastname: Crellin
authors.forename: Julian P
authors.initials: JP
authors.lastname: Vivian
authors.forename: Judith
authors.initials: J
authors.lastname: Scoble
authors.forename: Frances M
authors.initials: FM
authors.lastname: Chow
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: West
authors.forename: Rajini
authors.initials: R
authors.lastname: Brammananth
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Proellocks
authors.forename: Adam
authors.initials: A
authors.lastname: Shahine
authors.forename: Jerome
authors.initials: J
authors.lastname: Le Nours
authors.forename: Matthew C J
authors.initials: MC
authors.lastname: Wilce
authors.forename: Warwick J
authors.initials: WJ
authors.lastname: Britton
authors.forename: Ross L
authors.initials: RL
authors.lastname: Coppel
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.forename: Travis
authors.initials: T
authors.lastname: Beddoe
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D048230:Corynebacterium glutamicum / Q000201:enzymology; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D007783:Lactones / Q000737:chemistry* / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D000077403:Orlistat; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007783:Lactones; D000077403:Orlistat; D008049:Lipase