Open AccessIdentification, Isolation and Biochemical Characterization of a Phosphopantetheine:Protein Transferase that Activates the Two Type‐I Fatty Acid Synthases of Brevibacterium Ammoniagenes
Author(s) -
Stuible HansPeter,
Meier Sandra,
Schweizer Eckhart
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00481.x
Subject(s) - acyl carrier protein , biology , biochemistry , escherichia coli , microbiology and biotechnology , gene , brevibacterium , fatty acid synthase , heterologous expression , genetics , recombinant dna , microorganism , bacteria
Upon heterologous expression of the Brevibacterium ammoniagenes type‐I fatty acid synthase FAS‐A in Escherichia coli , only the pantetheine‐free apoenzyme is synthesized. Activation of FAS‐A to its holoform was achieved by transformation with a second B. ammoniagenes gene, PPTI , encoding a type‐I FAS‐specific phosphopantetheine transferase. PPTI was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to this linkage, PPT1 was part of the cloned fasB DNA region and, consequently, FAS‐B but not FAS‐A was synthesized as holoFAS in E. coli. PPTI encodes a protein of 153 amino acids and has a calculated molecular mass of 16884 Da. The PPT1 gene product contains 25% identical and 42 % conserved amino acids compared with the type‐II acyl‐carrier‐protein‐ activating enzyme of E. coli. Although there is essentially no intergenic region between jusB and PPT1 , the PPTase gene is autonomously expressed in E. coli if flanked by 200 bp of its endogenous 5′ DNA. The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS‐B. Overexpression and purification of the His‐tagged Ppt1p allowed the in vitro activation of apoFAS‐A. This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg 2+ and leads to a specific FAS activity comparable to that of natural B. ammoniugenes FAS‐A. The reactivity of the in vitro ‐activated FAS‐A was verified by the optical FAS assay and by analysis of its in vitro products. In agreement with the known overall colinearity of B. ammoniagenes FAS‐B and the Saccharomyces cerevisiae FAS1 and FAS2 gene products, a PPT1 ‐like sequence is also observed at the C terminus of FAS2. However, in contrast to B. ammioniagenes PPT1 , this sequence is an integral part of the yeast FAS2 gene. Thus, activation of type‐I fatty acid synthases may be accomplished by distinct trans ‐acting PPTase enzymes and by intrinsic cis ‐acting PPTase domains.