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. 1997 Jun;179(11):3697–3705. doi: 10.1128/jb.179.11.3697-3705.1997

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

L Tang 1, A C Weissborn 1, E P Kennedy 1
PMCID: PMC179167  PMID: 9171419

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 membrane-derived 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 gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.

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Selected References

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  1. Abita J. P., Lazdunski M., Ailhaud G. Structure-function relationships of the acyl-carrier protein of Escherichia coli. Eur J Biochem. 1971 Dec 10;23(3):412–420. doi: 10.1111/j.1432-1033.1971.tb01635.x. [DOI] [PubMed] [Google Scholar]
  2. Breedveld M. W., Miller K. J. Cyclic beta-glucans of members of the family Rhizobiaceae. Microbiol Rev. 1994 Jun;58(2):145–161. doi: 10.1128/mr.58.2.145-161.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cronan J. E., Jr, Narasimhan M. L., Rawlings M. Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes. Gene. 1988 Oct 15;70(1):161–170. doi: 10.1016/0378-1119(88)90114-x. [DOI] [PubMed] [Google Scholar]
  4. Frederick A. F., Kay L. E., Prestegard J. H. Location of divalent ion sites in acyl carrier protein using relaxation perturbed 2D NMR. FEBS Lett. 1988 Sep 26;238(1):43–48. doi: 10.1016/0014-5793(88)80222-9. [DOI] [PubMed] [Google Scholar]
  5. Hancock W. S., Marshall G. R., Vagelos P. R. Acyl carrier protein. XX. Chemical synthesis and characterization of analogues of acyl carrier protein. J Biol Chem. 1973 Apr 10;248(7):2424–2434. [PubMed] [Google Scholar]
  6. Heaton M. P., Neuhaus F. C. Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid. J Bacteriol. 1994 Feb;176(3):681–690. doi: 10.1128/jb.176.3.681-690.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Holak T. A., Kearsley S. K., Kim Y., Prestegard J. H. Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations. Biochemistry. 1988 Aug 9;27(16):6135–6142. doi: 10.1021/bi00416a046. [DOI] [PubMed] [Google Scholar]
  8. Hopwood D. A., Sherman D. H. Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet. 1990;24:37–66. doi: 10.1146/annurev.ge.24.120190.000345. [DOI] [PubMed] [Google Scholar]
  9. Issartel J. P., Koronakis V., Hughes C. Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation. Nature. 1991 Jun 27;351(6329):759–761. doi: 10.1038/351759a0. [DOI] [PubMed] [Google Scholar]
  10. Jackowski S., Rock C. O. Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants. J Bacteriol. 1987 Apr;169(4):1469–1473. doi: 10.1128/jb.169.4.1469-1473.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Katsube T., Kazuta Y., Tanizawa K., Fukui T. Expression in Escherichia coli of UDP-glucose pyrophosphorylase cDNA from potato tuber and functional assessment of the five lysyl residues located at the substrate-binding site. Biochemistry. 1991 Sep 3;30(35):8546–8551. doi: 10.1021/bi00099a008. [DOI] [PubMed] [Google Scholar]
  12. Kazuta Y., Omura Y., Tagaya M., Nakano K., Fukui T. Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals. Biochemistry. 1991 Sep 3;30(35):8541–8545. doi: 10.1021/bi00099a007. [DOI] [PubMed] [Google Scholar]
  13. Kennedy E. P. Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1092–1095. doi: 10.1073/pnas.79.4.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim Y., Prestegard J. H. A dynamic model for the structure of acyl carrier protein in solution. Biochemistry. 1989 Oct 31;28(22):8792–8797. doi: 10.1021/bi00448a017. [DOI] [PubMed] [Google Scholar]
  15. Kim Y., Prestegard J. H. Refinement of the NMR structures for acyl carrier protein with scalar coupling data. Proteins. 1990;8(4):377–385. doi: 10.1002/prot.340080411. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Loubens I., Debarbieux L., Bohin A., Lacroix J. M., Bohin J. P. Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae. Mol Microbiol. 1993 Oct;10(2):329–340. doi: 10.1111/j.1365-2958.1993.tb01959.x. [DOI] [PubMed] [Google Scholar]
  18. Majerus P. W. Acyl carrier protein: effects of acetylation and tryptic hydrolysis on function in fatty acid synthesis. Science. 1968 Jan 26;159(3813):428–430. doi: 10.1126/science.159.3813.428. [DOI] [PubMed] [Google Scholar]
  19. Olsen G. J., Woese C. R., Overbeek R. The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol. 1994 Jan;176(1):1–6. doi: 10.1128/jb.176.1.1-6.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Picard V., Ersdal-Badju E., Lu A., Bock S. C. A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 1994 Jul 11;22(13):2587–2591. doi: 10.1093/nar/22.13.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Platt M. W., Miller K. J., Lane W. S., Kennedy E. P. Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti. J Bacteriol. 1990 Sep;172(9):5440–5444. doi: 10.1128/jb.172.9.5440-5444.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rawlings M., Cronan J. E., Jr The gene encoding Escherichia coli acyl carrier protein lies within a cluster of fatty acid biosynthetic genes. J Biol Chem. 1992 Mar 25;267(9):5751–5754. [PubMed] [Google Scholar]
  23. Rock C. O., Cronan J. E., Jr Acyl-acyl carrier protein synthetase from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):163–168. doi: 10.1016/0076-6879(81)71023-1. [DOI] [PubMed] [Google Scholar]
  24. Schulman H., Kennedy E. P. Localization of membrane-derived oligosaccharides in the outer envelope of Escherichia coli and their occurrence in other Gram-negative bacteria. J Bacteriol. 1979 Jan;137(1):686–688. doi: 10.1128/jb.137.1.686-688.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  26. Shen Z., Byers D. M. Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis. J Bacteriol. 1996 Jan;178(2):571–573. doi: 10.1128/jb.178.2.571-573.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Spaink H. P., Lugtenberg B. J. Role of rhizobial lipo-chitin oligosaccharide signal molecules in root nodule organogenesis. Plant Mol Biol. 1994 Dec;26(5):1413–1422. doi: 10.1007/BF00016482. [DOI] [PubMed] [Google Scholar]
  28. Talaga P., Fournet B., Bohin J. P. Periplasmic glucans of Pseudomonas syringae pv. syringae. J Bacteriol. 1994 Nov;176(21):6538–6544. doi: 10.1128/jb.176.21.6538-6544.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Therisod H., Kennedy E. P. The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8235–8238. doi: 10.1073/pnas.84.23.8235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Therisod H., Weissborn A. C., Kennedy E. P. An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7236–7240. doi: 10.1073/pnas.83.19.7236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vanaman T. C., Wakil S. J., Hill R. L. The complete amino acid sequence of the acyl carrier protein of Escherichia coli. J Biol Chem. 1968 Dec 25;243(24):6420–6431. [PubMed] [Google Scholar]
  32. Weissborn A. C., Kennedy E. P. Biosynthesis of membrane-derived oligosaccharides. Novel glucosyltransferase system from Escherichia coli for the elongation of beta 1----2-linked polyglucose chains. J Biol Chem. 1984 Oct 25;259(20):12644–12651. [PubMed] [Google Scholar]
  33. Weissborn A. C., Rumley M. K., Kennedy E. P. Biosynthesis of membrane-derived oligosaccharides. Membrane-bound glucosyltransferase system from Escherichia coli requires polyprenyl phosphate. J Biol Chem. 1991 May 5;266(13):8062–8067. [PubMed] [Google Scholar]
  34. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van Golde L. M. Metabolism of membrane phospholipids and its relation to a novel class of oligosaccharides in Escherichia coli. Proc Natl Acad Sci U S A. 1973 May;70(5):1368–1372. doi: 10.1073/pnas.70.5.1368. [DOI] [PMC free article] [PubMed] [Google Scholar]

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