The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.
Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.
Schroeder
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BG
Tuberculosis Research Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Disease, National Institutes of Health, Twinbrook II, Room 239, 12441 Parklawn Drive, Rockville, Maryland 20852, USA.
Barry
C E
CE
D000085:Acetates / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009171:Mycolic Acids / Q000378:metabolism*; D013379:Substrate Specificity
D000085:Acetates; D009171:Mycolic Acids; D008780:Methyltransferases