IDENTIFICATION, ISOLATION AND BIOCHEMICAL-CHARACTERIZATION OF A PHOSPHOPANTETHEINE-PROTEIN TRANSFERASE THAT ACTIVATES THE 2 TYPE-I FATTY-ACID SYNTHASES OF BREVIBACTERIUM-AMMONIAGENES

Upon heterologous expression of the Brevibacterium ammoniagenes type-I fatty acid synthase FAS-A in Escherichia coli, only the pantetheine-f ree apoenzyme is synthesized. Activation of FAS-A to its holoform was achieved by transformation with a second B. ammoniagenes gene, PPT1, e ncoding a type-I FAS-specific phosphopantetheine transferase. PPT1 was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to t his linkage, PPT1 was part of the cloned fasB DNA region and, conseque ntly, FAS-B but not FAS-A was synthesized as holoFAS in E. coli. PPT1 encodes a protein of 153 amino acids and has a calculated molecular ma ss of 16884 Da. The PPT1 gene product contains 25% identical and 42% c onserved amino acids compared with the type-II acyl-carrier-protein-ac tivating enzyme of E. coli. Although there is essentially no intergeni c region between fasB and PPT1, the PPTase gene is autonomously expres sed in E. coli if flanked by 200 bp of its endogenous 5' DNA. The stru ctural independence of Ppt1p was confined immunologically, as specific antibodies react with the purified PPTase but not with FAS-B. Overexp ression and purification of the His-tagged Ppt1p allowed in in vitro a ctivation of apoFAS-A. This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg2+ and leads to a specific FAS activity comparabl e to that of natural B. ammoniagenes FAS-A. The reactivity of the in v itro-activated FAS-A was verified by the optical FAS assay and by anal ysis of its in vitro products. In agreement with the known overall col inearity of B. ammoniagenes FAS-B and the Saccharomyces cerevisiae FAS 1 and FAS2 gene products, a PPT1-like sequence is also observed at the C terminus of FAS2. However, in contrast to B. ammoniagenes PPT1, thi s sequence is an integral part of the yeast FAS2 gene. Thus, activatio n of type-I fatty acid synthase may be accomplished by distinct trans- acting PPTase enzymes and by intrinsic cis-acting PPTase domains.