Abstract
ThefabB gene of E. coli encoding β-ketoacyl-ACP synthase I has been isolated by complementation and sequenced. The enzyme has been purified and its NH2-terminal residues sequenced. Identification of the active site was accomplished by tagging with3H-cerulenin and radio sequencing of the region. Comparison of the deduced primary structures of thefabB gene product with theFAS2 gene product of Saccharomyces cerevisiae revealed the probable active site in chalcone synthases of higher plants.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- ACP:
-
acyl carrier protein
- bp:
-
base pair(s)
- EDTA:
-
ethylene diamine tetraacetic acid
- kb:
-
kilobase (pairs)
- nt:
-
nucleotide(s)
- ORF:
-
open reading frame
- PAGE:
-
polyacrylamide gel electrophoresis
- RNase:
-
ribonuclease
- SDS:
-
sodium dodecyl sulfate
- Tris:
-
tris-(hydroxymethyl)-aminomethane
References
Alberts, A. W., R. M. Bell &P. R. Vagelos: Acyl carrier protein. XV. Studies of β-ketoacyl-acyl carrier protein synthetase. J. Biol. Chem. 247, 3190–3198 (1972)
Becerril, B., F. Valle, E. Merino, L. Riba &F. Bolivar: Repetitive extragenic palindromic (REP) sequences in theEscherichia coli gdhA gene. Gene 37, 53–62 (1985)
Benton, W. D. &R. W. Davis: Screening λgt recombinant clones by hybridization to single plaquesin situ. Science 196, 180–182 (1977)
Birnboim, H. C. &J. Doly: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523 (1979)
Buttke, T. M. &L. O. Ingram: Inhibition of unsaturated fatty acid synthesis inEscherichia coli by the antibiotic cerulenin. Biochemistry 17, 5282–5286 (1978)
Clark, D. &J. E. Cronan Jr: Further mapping of several membrane lipid biosynthetic genes (fabC, fabB, gpsA, plsB) ofEscherichia coli. J. Bacteriol. 132, 549–554 (1977)
Cronan, J. E. Jr.,W.-B. Li, R. Coleman, M. Narasimhan, D. de Mendoza &J. M. Schwab: Derived amino acid sequence and identification of active site residues ofEscherichia coli β-hydroxydecanoyl thioester dehydrase. J. Biol. Chem. 263, 4641–4646 (1988)
D’Agnolo, G., I. S. Rosenfeld, J. Awaya, S. Ōmura &P. R. Vagelos: Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of β-ketoacyl-acyl carrier protein synthetase. Biochim. Biophys. Acta 326, 155–166 (1973)
D’Agnolo, G., I. S. Rosenfeld &P. R. Vagelos: Multiple forms of β-ketoacyl-acyl carrier protein synthetase inEscherichia coli. J. Biol. Chem. 250, 5289–5294 (1975)
Dimroth, P., H. Walter &F. Lynen: Biosynthese von 6-methylsalicylsäure. Eur. J. Biochem. 13, 98–110 (1970)
Dimroth, P., E. Ringelmann &F. Lynen: 6-methylsalicylic acid synthetase from Penicillium patulum. Some catalytic properties of the enzyme and its relation to fatty acid synthetase. Eur. J. Biochem. 68, 591–596 (1976)
Doolittle, R. F.: Protein evolution. In: The Proteins. H. Neurath, R. L. Hill and C.-L. Boeder, eds., Acad. Press, New York IV, 1–118 (1979)
Drawert, F. &J. Beier: Einbau von (14C)-essigsäure in hopfenbitterstoffe. Phytochemistry 13, 2749–2753 (1974)
Feinbaum, R. L. &F. M. Ausubel: Transcriptional regulation of theArabidopsis thaliana chalcone synthase gene. Mol. Cell Biol. 8, 1985–1992 (1988)
Funabashi, H., S. Iwasaki &S. Okuda: A model study on the mechanism of fatty acid synthetase inhibition by antibiotic cerulenin. Tetrahedron Lett. 24, 2673–2676 (1983)
Garwin, J. L., A. L. Klages &J. E. Cronan Jr: β-ketoacyl acyl carrier protein synthase II ofEscherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. J. Biol. Chem. 255, 3263–3265 (1980)
Garwin, J. L., A. L. Klages &J. E. Cronan Jr: Structural, enzymatic, and genetic studies of β-ketoacyl-acyl carrier protein synthases I and II ofEscherichia coli. J. Biol. Chem. 255, 11949–11956 (1980)
Gilman, M. Z. &M. J. Chamberlin: Developmental and genetic regulation of Bacillus subtilis genes transcribed by ϱ-28-RNA polymerase. Cell 35, 285–293 (1983)
Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B. Swebilius Singer &G. Stormo: Translational initiation in prokaryotes. Ann. Rev. Microbiol. 35, 365–403 (1981)
Greenspan, M. D., A. W. Alberts &P. R. Vagelos: Acyl carrier protein. XIII. β-ketoacyl acyl carrier protein synthetase fromEscherichia coli. J. Biol. Chem. 244, 6477–6485 (1969)
Hanahan, D.: Techniques for transformation ofE. coli. In: DNA Cloning, vol. 1, a practical approach. D. M. Glover, ed., IRL Press, Oxford, pp. 109–135 (1985)
Hattori, M. &Y. Sakaki: Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152, 232–238 (1986)
Hawley, D. K. &W. R. McClure: Compilation and analysis ofEscherichia coli promoter DNA sequences. Nucleic Acids Res. 11, 2237–2255 (1983)
Henikoff, S.: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351–359 (1984)
Herendeen, S. L., R. A. Van Bogelen &F. C. Neidhardt: Levels of major proteins ofEscherichia coli during growth at different temperatures. J. Bacteriol. 139, 185–194 (1979)
Higgins, C. F., G. F.-L. Ames, W. M. Barnes, J. M. Clement &M. Hofnung: A novel intercistronic regulatory element of prokaryotic operons. Nature 298, 760–762 (1982)
Jackowski, S. &C. O. Rock: Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria. J. Biol. Chem. 262, 7927–7931 (1987)
Kaiser, K. &N. E. Murray: The use of phage lambda replacement vectors in the construction of representative genomic DNA libraries. In: DNA Cloning vol. 1, a practical approach. D. M. Glover, ed., IRL Press, Oxford, pp. 1–47 (1987)
Kawaguchi, A., H. Tomoda, S. Nozoe, S. Ōmura &S. Okuda: Mechanism of action of cerulenin on fatty acid synthetase. Effect of cerulenin on iodoacetamide-induced malonyl-CoA decarboxylase activity. J. Biochem. 92, 7–12 (1982)
Koes, R. E., C. E. Spelt, H. J. Reif, P. J. van den Elzen, E. Veltkamp &J. N. Mol: Floral tissue ofPetunia hybrida (V 30) expresses only one member of the chalcone synthase multigene family. Nucleic Acids Res. 14, 5229–5239 (1986)
Koes, R. E.: Genes involved in flavonoid biosynthesis in Petunia hybrida: The chalcone synthase multigene family. Academisch proefschrift, Vrije Universiteit te Amsterdam 1988
Kohara, Y., K. Akiyama &K. Isono: The physical map of the whole E. coli chromosome: Application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50, 495–508 (1987)
Kresze, G.-B., L. Steber, D. Oesterhelt &F. Lynen: Reaction of yeast fatty acid synthetase with iodoacetamide. 2. Identification of the amino acid residues reacting with iodoacetamide and primary structure of a peptide containing the peripheral sulfhydryl group. Eur. J. Biochem. 79, 181–190 (1977)
Kresze, G.-B., L. Steber, D. Oesterhelt &F. Lynen: Reaction of yeast fatty acid synthetase with iodoacetamide. 3. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide. Eur. J. Biochem. 79, 191–199 (1977)
Kreuzaler, F. &K. Hahlbrock: Enzymic synthesis of an aromatic ring from acetate units. Partial purification and some properties of flavanone synthase from cell-suspension cultures ofPetroselinum hortense. Eur. J. Biochem. 56, 205–213 (1975)
Kreuzaler, F., R. J. Light &K. Hahlbrock: Flavanone synthase catalyzes CO2 exchange and decarboxylation of malonyl-CoA. FEBS Lett. 94, 175–178 (1978)
Kreuzaler, F., H. Ragg, W. Heller, R. Tesch, I. Witt, D. Hammer &K. Hahlbrock: Flavanone synthase fromPetroselinum hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue. Eur. J. Biochem. 99, 89–96 (1979)
Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)
Maniatis, T., E. F. Fritsch &J. Sambrook: Molecular cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1982)
Martin, J. F. &L. E. McDaniel: Specific inhibition of candicidin biosynthesis by the lipogenic inhibitor cerulenin. Biochim. Biophys. Acta 411, 186–194 (1975)
Matsumae, A., Y. Kamio & T. Hata: Studies on cerulenin. I. Studies on cerulenin producing strain. J. Antibiot. Ser. A, 236–238 (1963)
Mendoza, D. de, A. K. Ulrich &J. E. Cronan Jr: Thermal regulation of membrane fluidity inEscherichia coli. Effects of overproduction of β-ketoacyl-acyl carrier protein synthase I. J. Biol. Chem. 258, 2098–2101 (1983)
Messing, J.: New M13 vectors for cloning. Methods Enzymol. 101, 20–75 (1983)
Miura, K.: Preparation of bacterial DNA by the phenol-pH 9-RNases method. Methods Enzymol. XIIA, 543–545 (1967)
Murray, N. E., W. J. Brammer &K. Murray: Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150, 53–61 (1977)
Niesbach-Klösgen, U., E. Barzen, J. Bernhardt, W. Rohde, Z. Schwarz-Sommer, H. J. Reif, U. Wienand &H. Saedler: Chalcone synthase genes in plants: A tool to study evolutionary relationships. J. Mol. Evol. 26, 213–225 (1987)
Platt, T.: Transcription termination and the regulation of gene expression. Ann. Rev. Biochem. 55, 339–372 (1986)
Queen, C. &L. J. Korn: A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 12, 581–599 (1984)
Reimold, U., M. Kröger, F. Kreuzaler &K. Hahlbrock: Coding and 3′ non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme. EMBO J. 2, 1801–1805 (1983)
Rosenberg, M. &D. Court: Regulatory sequences involved in the promotion and termination of RNA transcription. Ann. Rev. Genet. 13, 319–353 (1979)
Ryder, T. B., S. A. Hedrick, J. N. Beli, X. Liang, S. D. Clouse &C. J. Lamb: Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase inPhaseolus vulgaris. Mol. Gen. Genet. 210, 219–233 (1987)
Sanger, F., S. Nicklen &A. R. Coulson: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467 (1977)
Scheuerbrandt, G. &K. Bloch: Unsaturated fatty acids in microorganisms. J. Biol. Chem. 237, 2064–2068 (1962)
Schweizer, E., G. Müller, L. M. Roberts, M. Schweizer, J. Rösch, P. Wiesner, J. Beck, D. Stratmann &I. Zauner: Genetic control of fatty acid synthetase biosynthesis and structure in lower fungi. Fat. Sci. Technol. 89, 570–577 (1987)
Schröder, G., J. W. Brown &J. Schröder: Molecular analysis of resveratrol synthase. cDNA, genomic clones and relationship with chalcone synthase. Eur. J. Biochem. 172, 161–169 (1988)
Schöppner, A. &H. Kindl: Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut. J. Biol. Chem. 259, 6806–6811 (1984)
Sharp, P. M. &W.-H. Li: Codon usage in regulatory genes inEscherichia coli does not reflect selection for ‘rare’ codons. Nucleic Acids Res. 14, 7737–7749 (1986)
Shine, J. &L. Dalgarno: The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342–1346 (1974)
Siggaard-Andersen, M.: Role of Escherichia coli β-ketoacyl-ACP synthase I in unsaturated fatty acid synthesis. Carlsberg Res. Commun. 53, 371–379 (1988)
Silbert, D. F.: Genetic modification of membrane lipid. Ann. Rev. Biochem. 44, 315–339 (1975)
Sommer, H. &H. Saedler: Structure of the chalcone synthase gene of Antirrhinum majus. Mol. Gen. Genet. 202, 429–434 (1986)
Stern, M. J., G. F.-L. Ames, N. H. Smith, E. C. Robinson &C. F. Higgins: Repetitive extragenic palindromic sequences: A major component of the bacterial genome. Cell 37, 1015–1026 (1984)
Tabor, S. &C. C. Richardson: DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767–4771 (1987)
Tomoda, H., A. Kawaguchi, S. Ōmura &S. Okuda: Cerulenin resistance in a cerulenin-producing fungus. II. Characterization of fatty acid synthetase fromCephalosporium caerulens. J. Biochem. 95, 1705–1712 (1984)
Tomoda, H., A. Kawaguchi, T. Yasuhara, T. Nakajima, S. Ōmura &S. Okuda: Cerulenin resistance in a cerulenin-producing fungus. III. Studies on active-site peptides of fatty acid synthetase fromCephalosporium caerulens. J. Biochem. 95, 1713–1723 (1984)
Toomey, R. E. &S. J. Wakil: Studies on the mechanism of fatty acid synthesis. XIV. Preparation and general properties of acyl-malonyl acyl carrier protein condensing enzyme fromEscherichia coli. J. Biol. Chem. 241, 1159–1165 (1966)
Vance, D., I. Goldberg, O. Mitsuhashi, K. Bloch, S. Ōmura &S. Nomura: Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochem. Biophys. Res. Commun. 48, 649–656 (1972)
Vogel, H. J. &D. M. Bonner: Acetylornithase of Escherichia coli: Partial purification and some properties. J. Biol. Chem. 218, 97–106 (1956)
Wettstein-Knowles, P. von: Role ofcer-cqu in epicuticular wax biosynthesis. Biochem. Soc. Trans. 14, 576–579 (1986)
Author information
Authors and Affiliations
Additional information
Accepted byS. O. Andersen
Rights and permissions
About this article
Cite this article
Kauppinen, S., Siggaard-Andersen, M. & von Wettstein-Knowles, P. β-ketoacyl-ACP synthase I of Escherichia coli: Nucleotide sequence of thefabB gene and identification of the cerulenin binding residue. Carlsberg Res. Commun. 53, 357–370 (1988). https://doi.org/10.1007/BF02983311
Issue Date:
DOI: https://doi.org/10.1007/BF02983311