Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis

Abstract

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membranederived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup ␥-3 have periplasmic glucans synthesized by the mechanism found in E. coli.Acyl carrier protein of Escherichia coli is a small (77-aminoacid) acidic protein that plays an essential role in several biosynthetic pathways: fatty acid biosynthesis (reviewed in reference 39), phospholipid biosynthesis (reviewed in reference 3), lipopolysaccharide biosynthesis (reviewed in reference 28), and activation of prohemolysin (11). All of these functions require the presence of the phosphopantetheine prosthetic group on serine 36 of acyl carrier protein. The terminal thiol on this prosthetic grou...

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

“…Leaves were infected with 10 7 CFU of WT D. dadantii, ⌬opgG mutant, or ⌬opgG mutant harboring pNF400 (ϩopgGH Ypst ). *, P Ͻ 0.05. affect the protein's ability to participate in OPG biosynthesis (14). Hence, our inability to detect any OPGs in Y. pseudotuberculosis might have been caused by a nonfunctional ACP.…”

“…S1 in the supplemental material). The latter operon is found in many gammaproteobacteria and is functionally homologous to the nvbAB, chvAB, and cgs loci found in alphaproteobacteria (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The opgGH operon encodes glucosyltransferases that synthesize branched glucans from UDP glucose (UDP-Glc), using an acyl carrier protein (ACP) as a cofactor (28,29).…”

Topological analysis of the membrane-bound glucosyltransferase, MdoH, required for osmoregulated periplasmic glucan synthesis in Escherichia coli

“…The three large cytoplasmic domains (C1, C2, and C5) are probably involved in the polymerization of glucose units from UDP-glucose. One can also expect that at least one of these domains interacts with the acyl carrier protein, whose exact function in the polymerization remains obscure (11,30,31), even though a recent study has shed light on the domains of this protein that are probably implicated in the interaction with MdoH (28).…”

Rhizobial acyl carrier proteins and their roles in the formation of bacterial cell-surface components that are required for the development of nitrogen-fixing root nodules on legume hosts

“…For this latter function, the prosthetic phosphopantetheine group, essential for lipid biosynthesis, is not needed. However, certain regions of the E. coli AcpP, conserved in other members of the γ‐3 subgroup of the proteobacteria, are required for its functioning in MDO biosynthesis [4]. The fact that such a small protein like AcpP is involved in two totally different functions questions the widely held biochemical dogma that one protein or protein domain might be responsible for one function only.…”