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. 1993 Apr;175(7):1865–1870. doi: 10.1128/jb.175.7.1865-1870.1993

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

D Fice 1, Z Shen 1, D M Byers 1
PMCID: PMC204243  PMID: 8384617

Abstract

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.

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Selected References

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  1. Anderson M. S., Raetz C. R. Biosynthesis of lipid A precursors in Escherichia coli. A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. J Biol Chem. 1987 Apr 15;262(11):5159–5169. [PubMed] [Google Scholar]
  2. Black P. N. Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport. J Bacteriol. 1991 Jan;173(2):435–442. doi: 10.1128/jb.173.2.435-442.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black P. N., Said B., Ghosn C. R., Beach J. V., Nunn W. D. Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli. J Biol Chem. 1987 Jan 25;262(3):1412–1419. [PubMed] [Google Scholar]
  4. Byers D. M. Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi. J Bacteriol. 1989 Jan;171(1):59–64. doi: 10.1128/jb.171.1.59-64.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Byers D. M., Holmes C. G. A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi. Biochem Cell Biol. 1990 Jul-Aug;68(7-8):1045–1051. doi: 10.1139/o90-154. [DOI] [PubMed] [Google Scholar]
  6. Byers D. M., Meighen E. A. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6085–6089. doi: 10.1073/pnas.82.18.6085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper C. L., Hsu L., Jackowski S., Rock C. O. 2-Acylglycerolphosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein. J Biol Chem. 1989 May 5;264(13):7384–7389. [PubMed] [Google Scholar]
  8. Cronan J. E., Jr Evidence that incorporation of exogenous fatty acids into the phospholipids of Escherichia coli does not require acyl carrier protein. J Bacteriol. 1984 Aug;159(2):773–775. doi: 10.1128/jb.159.2.773-775.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. GERHART J. C., PARDEE A. B. The enzymology of control by feedback inhibition. J Biol Chem. 1962 Mar;237:891–896. [PubMed] [Google Scholar]
  10. Green P. R., Merrill A. H., Jr, Bell R. M. Membrane phospholipid synthesis in Escherichia coli. Purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase. J Biol Chem. 1981 Nov 10;256(21):11151–11159. [PubMed] [Google Scholar]
  11. Gunsalus-Miguel A., Meighen E. A., Nicoli M. Z., Nealson K. H., Hastings J. W. Purification and properties of bacterial luciferases. J Biol Chem. 1972 Jan 25;247(2):398–404. [PubMed] [Google Scholar]
  12. Hsu L., Jackowski S., Rock C. O. Isolation and characterization of Escherichia coli K-12 mutants lacking both 2-acyl-glycerophosphoethanolamine acyltransferase and acyl-acyl carrier protein synthetase activity. J Biol Chem. 1991 Jul 25;266(21):13783–13788. [PubMed] [Google Scholar]
  13. 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]
  14. Jackowski S., Rock C. O. Transfer of fatty acids from the 1-position of phosphatidylethanolamine to the major outer membrane lipoprotein of Escherichia coli. J Biol Chem. 1986 Aug 25;261(24):11328–11333. [PubMed] [Google Scholar]
  15. Kameda K., Nunn W. D. Purification and characterization of acyl coenzyme A synthetase from Escherichia coli. J Biol Chem. 1981 Jun 10;256(11):5702–5707. [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. Mattoo A. K., Callahan F. E., Mehta R. A., Ohlrogge J. B. Rapid in Vivo Acylation of Acyl Carrier Protein with Exogenous Fatty Acids in Spirodela oligorrhiza. Plant Physiol. 1989 Feb;89(2):707–711. doi: 10.1104/pp.89.2.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Overath P., Pauli G., Schairer H. U. Fatty acid degradation in Escherichia coli. An inducible acyl-CoA synthetase, the mapping of old-mutations, and the isolation of regulatory mutants. Eur J Biochem. 1969 Feb;7(4):559–574. [PubMed] [Google Scholar]
  19. Ray T. K., Cronan J. E., Jr Activation of long chain fatty acids with acyl carrier protein: demonstration of a new enzyme, acyl-acyl carrier protein synthetase, in Escherichia coli. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4374–4378. doi: 10.1073/pnas.73.12.4374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rock C. O., Jackowski S. Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli. J Biol Chem. 1985 Oct 15;260(23):12720–12724. [PubMed] [Google Scholar]
  21. Rock C. O. Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli. J Biol Chem. 1984 May 25;259(10):6188–6194. [PubMed] [Google Scholar]
  22. Rodriguez A., Meighen E. Fatty acyl-AMP as an intermediate in fatty acid reduction to aldehyde in luminescent bacteria. J Biol Chem. 1985 Jan 25;260(2):771–774. [PubMed] [Google Scholar]
  23. Shen Z., Fice D., Byers D. M. Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase. Anal Biochem. 1992 Jul;204(1):34–39. doi: 10.1016/0003-2697(92)90135-t. [DOI] [PubMed] [Google Scholar]
  24. Silbert D. F., Ruch F., Vagelos P. R. Fatty acid replacements in a fatty acid auxotroph of Escherichia coli. J Bacteriol. 1968 May;95(5):1658–1665. doi: 10.1128/jb.95.5.1658-1665.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Silverstein E., Sulebele G. Modulation of heart muscle mitochondrial malate dehydrogenase activity. I. Activation and inhibition by p-mercuribenzoate. Biochemistry. 1970 Jan 20;9(2):274–282. doi: 10.1021/bi00804a013. [DOI] [PubMed] [Google Scholar]
  26. Soly R. R., Meighen E. A. Identification of the acyl transfer site of fatty acyl-protein synthetase from bioluminescent bacteria. J Mol Biol. 1991 May 5;219(1):69–77. doi: 10.1016/0022-2836(91)90858-4. [DOI] [PubMed] [Google Scholar]
  27. Ulitzur S., Hastings J. W. Myristic acid stimulation of bacterial bioluminescence in "aldehyde" mutants. Proc Natl Acad Sci U S A. 1978 Jan;75(1):266–269. doi: 10.1073/pnas.75.1.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wall L. A., Byers D. M., Meighen E. A. In vivo and in vitro acylation of polypeptides in Vibrio harveyi: identification of proteins involved in aldehyde production for bioluminescence. J Bacteriol. 1984 Aug;159(2):720–724. doi: 10.1128/jb.159.2.720-724.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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