In Vivo Functional Analyses of the Type II Acyl Carrier Proteins of Fatty Acid Biosynthesis

Abstract

Acyl carrier protein (ACP) is a key component of the fatty acid synthesis pathways of both type I and type II synthesis systems. A large number of structure-function studies of various type II ACPs have been reported, but all are in vitro studies that assayed function or interaction of mutant ACPs with various enzymes of fatty acid synthesis or transfer. Hence in these studies functional properties of various mutant ACPs were assayed with only a subset of the many ACP-interacting proteins, which may not give an accurate overall view of the function of these proteins in vivo. This is especially so because Escherichia coli ACP has been reported to interact with several proteins that have no known roles in lipid metabolism. We therefore tested a large number of mutant derivatives of E. coli ACP carrying single amino acid substitutions for their abilities to restore growth to an E. coli strain carrying a temperature-sensitive mutation in acpP, the gene that encodes ACP. Many of these mutant proteins had previously been tested in vitro thus providing data for comparison with our results. We found that several mutant ACPs containing substitutions of ACP residues reported previously to be required for ACP function in vitro support normal growth of the acpP mutant strain. However, several mutant proteins reported to be severely defective in vitro failed to support growth of the acpP strain in vivo (or supported only weak growth). A collection of ACPs from diverse bacteria and from three eukaryotic organelles was also tested. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from two related bacteria, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth. Strikingly the ACP is that of the apicoplast of Plasmodium falciparum (the protozoan that causes malaria). The fact that an ACP from a such diverse organism can replace AcpP function in E. coli suggests that some of the protein-protein interactions detected for AcpP may be not be essential for growth of E. coli.Both type I and type II fatty acid biosynthetic systems have a key component, the acyl carrier protein (ACP), 2 that is essential for function (1-3). In both synthetic systems the ACP component carries fatty acyl intermediates among the different enzyme active sites. To perform this function ACP must be posttranslationally modified via the transfer of a 4Ј-phosphopantetheine moiety from CoA to a conserved serine residue of ACP. Attachment of this prosthetic group is mandatory because the thiol group of the phosphopantetheinyl moiety carries the intermediates of fatty acid biosynthesis. In the type I systems found in the mammalian cytosol, ACP is a domain of a very large polypeptide that contains all the enzymatic domains required for fatty acid biosynthesis (4). The functional mammalian fatty-acid synthase is a dimer of two identical polypeptides in head-to-head orientation that synthesizes the fatty acids used for complex lipid biosynthesis (5-7). The finished f...

The Conserved Modular Elements of the Acyl Carrier Proteins of Lipid Synthesis Are Only Partially Interchangeable

“…The lack of function of the L. lactis and E. faecalis AcpAs could be explained by the failure of these two highly similar ACPs to be modified by the E. coli AcpS 4Ј-phosphopantetheinyl transferase. However, upon co-expression with the promiscuous B. subtilis Sfp 4Ј-phosphopantetheinyl transferase, both AcpA proteins were modified but remained unable to restore growth to a conditionally lethal acpP mutant strain of E. coli (4). These findings were puzzling because we had previously observed that genes from both of these closely related bacteria could replace the functions of E. coli fatty acid genes in vivo (6 -8).…”

“…Escherichia coli ACP, the most thoroughly studied member of this protein family, is a small (77 residues; molecular weight, 8,860), very acidic (pI of 4.1), and extremely soluble protein. In prior work, we reported that ACP species encoded by 11 diverse bacteria plus that of the apicoplast of the malarial protozoan parasite Plasmodium falciparum functionally replaced E. coli ACP in vivo (4). This generality was unforeseen because E. coli ACP interacts with 21 different lipid metabolism enzymes (5), virtually all of which are essential for growth.…”

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

“…7A). Consistent with the finding that V. harveyi wild-type ACP is able to complement growth of an E. coli strain with a temperature-sensitive ACP (NRD224) at the restrictive temperature (35), expression of the linL46W protein led to strong complementation of the CY1861 strain in the presence of glucose and IPTG. Strikingly, expression of the cycL46W protein under similar conditions also led to complementation (Fig.…”

Bacteria Possessing Two RelA/SpoT-Like Proteins Have Evolved a Specific Stringent Response Involving the Acyl Carrier Protein-SpoT Interaction

“…3). It was shown previously that the acp Bsu gene compensated for the acpP(Ts) mutation in E. coli (6). This demonstrated again that fusion with the two-hybrid domain did not alter the function of the protein.…”