The biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene) transfer to acyl carrier protein bound meromycolic acid in vitro.
A closely related family of enzymes from Mycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-L-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain.
Tuberculosis Research Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840, USA.
Y
Y
Yuan
D
D
Mead
B G
BG
Schroeder
Y
Y
Zhu
C E
CE
Barry
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006838:Hydrocarbons; D008697:Methane / Q000031:analogs & derivatives* / Q000378:metabolism; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009171:Mycolic Acids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000213:Acyl Carrier Protein; D017931:DNA Primers; D006838:Hydrocarbons; D009171:Mycolic Acids; D011994:Recombinant Proteins; C013596:meromycolic acids; C030011:carbene; D008780:Methyltransferases; D008697:Methane