The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

No abstract

Holo-(acyl carrier protein) synthase (AcpS) posttranslationally modifies apoacyl carrier protein (apoACP) via transfer of 4-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 ␥-OH of apoACP. The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis. The acpS gene has previously been identified and shown to be essential for Escherichia coli growth. Earlier mutagenic studies isolated the E. coli MP4 strain, whose elevated growth requirement for CoA was ascribed to a deficiency in holoACP synthesis. Sequencing of the acpS gene from the E. coli MP4 strain (denoted acpS1) showed that the AcpS1 protein contains a G4D mutation. AcpS1 exhibited a ϳ5-fold reduction in its catalytic efficiency when compared with wild type AcpS, accounting for the E. coli MP4 strain phenotype. It is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar to the E. coli MP4 strain. In addition, it is demonstrated that the gene product, YhhU, of a previously identified E. coli open reading frame can completely suppress the acpS conditional, lethal phenotype upon overexpression of the protein, suggesting that YhhU may be involved in an alternative pathway for phosphopantetheinyl transfer and holoACP synthesis in E. coli.Escherichia coli utilizes a repeated cycle of condensation, reduction, dehydration, and isomerization reactions to produce saturated and unsaturated fatty acids (1, 2). This biosynthetic mechanism employs multiple enzymatic activities conglomerately termed the fatty acid synthase. The central coenzyme in all fatty acid synthases is the holo form of acyl carrier protein.ACPs, 1 as free proteins or as domains of large multifunctional proteins, function in a variety of synthases as hubs to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final compound. The apo to holo conversion of ACPs by post-translational modification with the 4Ј-phosphopantetheine (4Ј-PP) prosthetic group is essential to their function, since all acyl chain molecules undergoing biosynthesis by these systems are attached to the terminal cysteamine thiol of 4Ј-PP via a thioester bond.The E. coli fatty acid synthase has served as a model system for understanding the 4Ј-PP post-translational modification, structure, and function of ACPs. The E. coli ACP is a highly acidic protein of 77 residues. It is composed primarily of a 3-helix bundle (3, 4), and recent structural studies reinforce the notion that many ACPs from different synthases and/or organisms are of similar size and structure to E. coli ACP (5-7).E. coli ACP is post-translationally modified by holo-(acyl carrier protein) synthase (EC 2.7.8.7) in a Mg 2ϩ -dependent reaction, where the 4Ј-PP moiety is transferred from CoA to the conserved serine 36 ␥-OH of ACP (8). AcpS therefore converts inactive apoACP to its activated holo form. HoloACP is then capable of being a...