Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin.

摘要: Mycocerosyl lipids are found uniquely in the cell walls of pathogenic mycobacteria. Mycocerosic acid synthase (MAS) is a multifunctional protein which catalyzes elongation n-fatty acyl-CoA with methylamalonyl-CoA as elongating agent (Rainwater, D. L., and Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 616-623). To understand how various domains that catalyze reactions involved chain organized, mas gene from Mycobacterium tuberculosis bovis BCG was cloned. A lambda gt11 library AluI partially digested genomic DNA organism screened an oligonucleotide probe designed N-terminal amino sequence purified MAS. Using terminal segments inserts positive clones probe, rescreened process repeated. Sequencing four overlapping revealed contiguous 9699 base pair(s) (bp) mycobacterial genome containing 6330-bp open reading frame could code for 2100 acids molecular mass 225,437 daltons. The authenticity MAS verified by correspondence sequences deduced directly determined N terminus three different internal peptide fragments. By comparing active site regions known fatty synthases polyketide functional were identified. This analysis showed organized following order: beta-ketoacyl synthase, acyl transferase, dehydratase-enoyl reductase, beta-ketoreductase, carrier protein; no thioesterase-like domain be found. These results establish first case enzyme composed two identical subunits resemble vertebrate size, subunit structure, linear organization domains. Southern Western blot analyses absence encoded proteins smegmatis Escherichia coli. result consistent report mycocerosic present only