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. 1994 Apr 12;91(8):3044–3048. doi: 10.1073/pnas.91.8.3044

Yeast farnesyl-diphosphate synthase: site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II.

L Song 1, C D Poulter 1
PMCID: PMC43511  PMID: 8159703

Abstract

Prenyltransferases that catalyze the fundamental chain elongation reaction in the isoprenoid biosynthetic pathway contain several highly conserved amino acids, including two aspartate-rich regions thought to be involved in substrate binding and catalysis. We report a study of site-directed mutants for yeast farnesyl-diphosphate synthase (FPPSase; geranyl-diphosphate:isopentenyl-diphosphate, EC 2.5.1.10), a prenyltransferase that catalyzes the sequential 1'-4 coupling of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate and geranyl diphosphate. A recombinant form of FPPSase extended by a C-terminal -Glu-Glu-Phe alpha-tubulin epitope (EEF in single-letter amino acid code) was engineered to facilitate rapid purification of the enzyme by immunoaffinity chromatography and to remove traces of contaminating activity from wild-type FPPSase in the Escherichia coli host. Ten site-directed mutants were constructed in FPPSase::EEF. The six aspartates in domain I (at positions 100, 101, and 104) and domain II (at positions 240, 241, and 244) were changed to alanine (mutants designated D100A, D101A, D104A, D240A, D241A, and D244A); three arginine residues were changed, Arg-109 and Arg-110 to glutamine and Arg-350 to alanine (mutants designated R109Q, R110Q, and R350A); and Lys-254 was converted to alanine (mutant designated K254A). Mutations of the aspartatic residues and nearby arginine residues in domain I and Asp-240 and Asp-241 in domain II drastically lowered the catalytic activity of FPPSase::EEF. The D244A and K254A mutants were substantially less active, while kcat and the Michaelis constants for the R350A mutant were similar to those of FPPSase::EEF. Addition of an -EEF epitope to the C terminus of wild-type FPPSase resulted in a 14-fold increase of KmIPP and a 12-fold decrease of kcat, suggesting that the conserved hydrophilic C terminus of the enzyme may have a role in substrate binding and catalysis.

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Selected References

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  1. Anderson M. S., Yarger J. G., Burck C. L., Poulter C. D. Farnesyl diphosphate synthetase. Molecular cloning, sequence, and expression of an essential gene from Saccharomyces cerevisiae. J Biol Chem. 1989 Nov 15;264(32):19176–19184. [PubMed] [Google Scholar]
  2. Ashby M. N., Edwards P. A. Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase. J Biol Chem. 1990 Aug 5;265(22):13157–13164. [PubMed] [Google Scholar]
  3. Ashby M. N., Edwards P. A. Elucidation of the deficiency in two yeast coenzyme Q mutants. Characterization of the structural gene encoding hexaprenyl pyrophosphate synthetase. J Biol Chem. 1990 Aug 5;265(22):13157–13164. [PubMed] [Google Scholar]
  4. Barnard G. F., Langton B., Popják G. Pseudo-isoenzyme forms of liver prenyl transferase. Biochem Biophys Res Commun. 1978 Dec 14;85(3):1097–1103. doi: 10.1016/0006-291x(78)90655-1. [DOI] [PubMed] [Google Scholar]
  5. Barnard G. F., Popják G. Human liver prenyltransferase and its characterization. Biochim Biophys Acta. 1981 Sep 15;661(1):87–99. doi: 10.1016/0005-2744(81)90086-3. [DOI] [PubMed] [Google Scholar]
  6. Blanchard L., Karst F. Characterization of a lysine-to-glutamic acid mutation in a conservative sequence of farnesyl diphosphate synthase from Saccharomyces cerevisiae. Gene. 1993 Mar 30;125(2):185–189. doi: 10.1016/0378-1119(93)90326-x. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Brems D. N., Bruenger E., Rilling H. C. Isolation and characterization of a photoaffinity-labeled peptide from the catalytic site of prenyltransferase. Biochemistry. 1981 Jun 23;20(13):3711–3718. doi: 10.1021/bi00516a007. [DOI] [PubMed] [Google Scholar]
  9. Brems D. N., Rilling H. C. Photoaffinity labeling of the catalytic site of prenyltransferase. Biochemistry. 1979 Mar 6;18(5):860–864. doi: 10.1021/bi00572a019. [DOI] [PubMed] [Google Scholar]
  10. Chen A., Poulter C. D. Purification and characterization of farnesyl diphosphate/geranylgeranyl diphosphate synthase. A thermostable bifunctional enzyme from Methanobacterium thermoautotrophicum. J Biol Chem. 1993 May 25;268(15):11002–11007. [PubMed] [Google Scholar]
  11. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  12. Eberhardt N. L., Rilling H. C. Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties. J Biol Chem. 1975 Feb 10;250(3):863–866. [PubMed] [Google Scholar]
  13. Fujisaki S., Hara H., Nishimura Y., Horiuchi K., Nishino T. Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli. J Biochem. 1990 Dec;108(6):995–1000. doi: 10.1093/oxfordjournals.jbchem.a123327. [DOI] [PubMed] [Google Scholar]
  14. Fujisaki S., Nishino T., Katsuki H. Isoprenoid synthesis in Escherichia coli. Separation and partial purification of four enzymes involved in the synthesis. J Biochem. 1986 May;99(5):1327–1337. doi: 10.1093/oxfordjournals.jbchem.a135600. [DOI] [PubMed] [Google Scholar]
  15. Koyama T., Obata S., Osabe M., Takeshita A., Yokoyama K., Uchida M., Nishino T., Ogura K. Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: molecular cloning, sequence determination, overproduction, and purification. J Biochem. 1993 Mar;113(3):355–363. doi: 10.1093/oxfordjournals.jbchem.a124051. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Laskovics F. M., Poulter C. D. Prenyltransferase; determination of the binding mechanism and individual kinetic constants for farnesylpyrophosphate synthetase by rapid quench and isotope partitioning experiments. Biochemistry. 1981 Mar 31;20(7):1893–1901. doi: 10.1021/bi00510a027. [DOI] [PubMed] [Google Scholar]
  19. Marrero P. F., Poulter C. D., Edwards P. A. Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity. J Biol Chem. 1992 Oct 25;267(30):21873–21878. [PubMed] [Google Scholar]
  20. Matsuoka S., Sagami H., Kurisaki A., Ogura K. Variable product specificity of microsomal dehydrodolichyl diphosphate synthase from rat liver. J Biol Chem. 1991 Feb 25;266(6):3464–3468. [PubMed] [Google Scholar]
  21. Reed B. C., Rilling H. C. Crystallization and partial characterization of prenyltransferase from avian liver. Biochemistry. 1975 Jan 14;14(1):50–54. doi: 10.1021/bi00672a009. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sheares B. T., White S. S., Molowa D. T., Chan K., Ding V. D., Kroon P. A., Bostedor R. G., Karkas J. D. Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells. Biochemistry. 1989 Oct 3;28(20):8129–8135. doi: 10.1021/bi00446a025. [DOI] [PubMed] [Google Scholar]
  24. Skinner R. H., Bradley S., Brown A. L., Johnson N. J., Rhodes S., Stammers D. K., Lowe P. N. Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins. J Biol Chem. 1991 Aug 5;266(22):14163–14166. [PubMed] [Google Scholar]
  25. Stammers D. K., Tisdale M., Court S., Parmar V., Bradley C., Ross C. K. Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope. FEBS Lett. 1991 Jun 3;283(2):298–302. doi: 10.1016/0014-5793(91)80613-8. [DOI] [PubMed] [Google Scholar]
  26. Teruya J. H., Kutsunai S. Y., Spear D. H., Edwards P. A., Clarke C. F. Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA. Mol Cell Biol. 1990 May;10(5):2315–2326. doi: 10.1128/mcb.10.5.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weinstein J. D., Branchaud R., Beale S. I., Bement W. J., Sinclair P. R. Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells. Arch Biochem Biophys. 1986 Feb 15;245(1):44–50. doi: 10.1016/0003-9861(86)90188-8. [DOI] [PubMed] [Google Scholar]
  28. Yeh L. S., Rilling H. C. Purification and properties of pig liver prenyltransferase: interconvertible forms of the enzyme. Arch Biochem Biophys. 1977 Oct;183(2):718–725. doi: 10.1016/0003-9861(77)90405-2. [DOI] [PubMed] [Google Scholar]
  29. Zhang D., Jennings S. M., Robinson G. W., Poulter C. D. Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Arch Biochem Biophys. 1993 Jul;304(1):133–143. doi: 10.1006/abbi.1993.1331. [DOI] [PubMed] [Google Scholar]

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