Synthesis of mycolic acids of mycobacteria: an assessment of the cell-free system in light of the whole genome.

Mycolic acids are 70-90 carbon, alpha-alkyl, beta-hydroxy fatty acids constituting a major component of the cell envelope of Mycobacterium tuberculosis. The fact that the mycolic acid biosynthetic pathway is both essential in mycobacteria and the target for many first-line anti-TB drugs necessitates a detailed understanding of its biochemistry. A whole cell-free, but cell particulate- and membrane-containing enzyme preparation for mycolic acid biosynthesis was developed a few years ago and studied extensively. This system was shown to catalyze the synthesis of mature mycolic acids from [14C]acetate, but allows only minimal deposition into the cell wall proper. In the meantime the sequence of the entire genome of M. tuberculosis has been elucidated and its analysis using numerous protein sequence-based algorithms predicted cytoplasmic localization and a soluble, not a particulate, nature for the enzymes involved in the mycolic acid synthetic pathway. Accordingly, we re-assessed the 'cell-free' system for mycolic acid synthesis and concluded that it is probably due to the presence of unbroken cells, since viable cells were recovered from the cell wall preparation. The amount of whole cells depended upon the efficiency of the cell disruption method and conditions, and the amount of mycolic acid synthesized by the putative cell-free system correlated with the content of whole cells. Thus, accumulated results from the use of this 'cell-free' cell wall-based system should be re-evaluated in the light of these new data.