Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species.
Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.
Korduláková
Jana
J
Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Gilleron
Martine
M
Puzo
Germain
G
Brennan
Patrick J
PJ
Gicquel
Brigitte
B
Mikusová
Katarina
K
Jackson
Mary
M
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics*; D000483:Alleles; D015139:Blotting, Southern; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D050356:Lipid Metabolism; D008070:Lipopolysaccharides / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000737:chemistry; D008956:Models, Chemical; D009154:Mutation; D009161:Mycobacterium / Q000201:enzymology*; D020102:Mycobacterium smegmatis / Q000235:genetics; D016366:Open Reading Frames; D019308:Palmitic Acid / Q000737:chemistry / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D008070:Lipopolysaccharides; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; C050016:lipoarabinomannan; C064351:lipomannan; C034154:phosphatidylinositol mannoside; D019308:Palmitic Acid; D000217:Acyltransferases; D012697:Serine Endopeptidases; D008358:Mannose