Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Dec 15;288(Pt 3):753–757. doi: 10.1042/bj2880753

Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles.

A Chelstowska 1, T Zoladek 1, J Garey 1, J Kushner 1, J Rytka 1, R Labbe-Bois 1
PMCID: PMC1131950  PMID: 1471989

Abstract

The molecular basis of the uroporphyrinogen decarboxylase defect in eleven yeast 'uroporphyric' mutants was investigated. Uroporphyrinogen decarboxylase, an enzyme of the haem-biosynthetic pathway, catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen and is encoded by the HEM12 gene in the yeast Saccharomyces cerevisiae. The mutations were identified by sequencing the mutant hem12 alleles amplified in vitro from genomic DNA extracted from the mutant strains. Four mutations leading to the absence of enzyme protein were found: one mutation caused the substitution of the translation initiator Met to Ile, a two-base deletion created a frameshift at codon 247 and two nonsense mutations were found at codons 50 and 263. Four different point mutations were identified in seven 'leaky' mutants with residual modified uroporphyrinogen decarboxylase activity; each of three mutations was found in two independently isolated mutants. The nucleotide transitions resulted in the amino acid substitutions Ser-59 to Phe, Thr-62 to Ile, Leu-107 to Ser, or Ser-215 to Asn, all located in or near highly conserved regions. The results suggest that there is a single active centre in uroporphyrinogen decarboxylase, the geometry of which is affected in the mutant enzymes.

Full text

PDF
753

Images in this article

755Fig. 1.
on p.755
755Fig. 2.
on p.755

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arrese M. R., Vojensky D., Mattoon J. R. Characterization of a cytochrome oxidase-deficient yeast mutant which accumulates porphyrins. Biochem Biophys Res Commun. 1982 Aug;107(3):848–855. doi: 10.1016/0006-291x(82)90600-3. [DOI] [PubMed] [Google Scholar]
  2. Barnard G. F., Akhtar M. Stereochemical and mechanistic studies on the decarboxylation of uroporphyrinogen III in haem biosynthesis. J Chem Soc Perkin 1. 1979;10:2354–2360. doi: 10.1039/p19790002354. [DOI] [PubMed] [Google Scholar]
  3. Elder G. H., Evans J. O. A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver. Biochem J. 1978 Jan 1;169(1):205–214. doi: 10.1042/bj1690205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elder G. H., Tovey J. A., Sheppard D. M. Purification of uroporphyrinogen decarboxylase from human erythrocytes. Immunochemical evidence for a single protein with decarboxylase activity in human erythrocytes and liver. Biochem J. 1983 Oct 1;215(1):45–55. doi: 10.1042/bj2150045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Felix F., Brouillet N. Purification and properties of uroporphyrinogen decarboxylase from Saccharomyces cerevisiae. Yeast uroporphyrinogen decarboxylase. Eur J Biochem. 1990 Mar 10;188(2):393–403. doi: 10.1111/j.1432-1033.1990.tb15416.x. [DOI] [PubMed] [Google Scholar]
  6. Garey J. R., Hansen J. L., Harrison L. M., Kennedy J. B., Kushner J. P. A point mutation in the coding region of uroporphyrinogen decarboxylase associated with familial porphyria cutanea tarda. Blood. 1989 Mar;73(4):892–895. [PubMed] [Google Scholar]
  7. Garey J. R., Harrison L. M., Franklin K. F., Metcalf K. M., Radisky E. S., Kushner J. P. Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda. J Clin Invest. 1990 Nov;86(5):1416–1422. doi: 10.1172/JCI114856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Garey J. R., Labbe-Bois R., Chelstowska A., Rytka J., Harrison L., Kushner J., Labbe P. Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity. Eur J Biochem. 1992 May 1;205(3):1011–1016. doi: 10.1111/j.1432-1033.1992.tb16868.x. [DOI] [PubMed] [Google Scholar]
  9. Gollub E. G., Liu K. P., Dayan J., Adlersberg M., Sprinson D. B. Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene. J Biol Chem. 1977 May 10;252(9):2846–2854. [PubMed] [Google Scholar]
  10. Kawanishi S., Seki Y., Sano S. Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers. J Biol Chem. 1983 Apr 10;258(7):4285–4292. [PubMed] [Google Scholar]
  11. Kiel J. A., Boels J. M., Beldman G., Venema G. Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis. Gene. 1990 Apr 30;89(1):77–84. doi: 10.1016/0378-1119(90)90208-9. [DOI] [PubMed] [Google Scholar]
  12. Kurlandzka A., Rytka J. Mutants of Saccharomyces cerevisiae partially defective in the last steps of the haem biosynthetic pathway: isolation and genetical characterization. J Gen Microbiol. 1985 Nov;131(11):2909–2918. doi: 10.1099/00221287-131-11-2909. [DOI] [PubMed] [Google Scholar]
  13. Kurlandzka A., Zoladek T., Rytka J., Labbe-Bois R. The alternative pathway of haem synthesis via dehydroisocoproporphyrinogen in mutants of Saccharomyces cerevisiae partially deficient in uroporphyrinogen decarboxylase activity. Biochem J. 1991 Jan 1;273(Pt 1):246–247. doi: 10.1042/bj2730246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kurlandzka A., Zoladek T., Rytka J., Labbe-Bois R., Urban-Grimal D. The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker's yeast (Saccharomyces cerevisiae). Biochem J. 1988 Jul 1;253(1):109–116. doi: 10.1042/bj2530109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Labbe-Bois R., Rytka J., Litwinska J., Bilinski T. Analysis of heme biosynthesis in catalase and cytochrome deficient yeast mutants. Mol Gen Genet. 1977 Nov 14;156(2):177–183. doi: 10.1007/BF00283490. [DOI] [PubMed] [Google Scholar]
  16. Labbe-Bois R. The ferrochelatase from Saccharomyces cerevisiae. Sequence, disruption, and expression of its structural gene HEM15. J Biol Chem. 1990 May 5;265(13):7278–7283. [PubMed] [Google Scholar]
  17. Lash T. D. Action of uroporphyrinogen decarboxylase on uroporphyrinogen-III: a reassessment of the clockwise decarboxylation hypothesis. Biochem J. 1991 Sep 15;278(Pt 3):901–903. doi: 10.1042/bj2780901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Losson R., Lacroute F. Interference of nonsense mutations with eukaryotic messenger RNA stability. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5134–5137. doi: 10.1073/pnas.76.10.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luo J., Lim C. K. Decarboxylation of uroporphyrinogen III by erythrocyte uroporphyrinogen decarboxylase. Evidence for a random decarboxylation mechanism. Biochem J. 1990 Jun 1;268(2):513–515. doi: 10.1042/bj2680513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moore C. W., Hampsey D. M., Ernst J. F., Sherman F. Differential mismatch repair can explain the disproportionalities between physical distances and recombination frequencies of cyc1 mutations in yeast. Genetics. 1988 May;119(1):21–34. doi: 10.1093/genetics/119.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  22. Romana M., Le Boulch P., Roméo P. H. Rat uroporphyrinogen decarboxylase cDNA: nucleotide sequence and comparison to human uroporphyrinogen decarboxylase. Nucleic Acids Res. 1987 Sep 11;15(17):7211–7211. doi: 10.1093/nar/15.17.7211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Romeo P. H., Dubart A., Grandchamp B., de Verneuil H., Rosa J., Nordmann Y., Goossens M. Isolation and identification of a cDNA clone coding for rat uroporphyrinogen decarboxylase. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3346–3350. doi: 10.1073/pnas.81.11.3346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roméo P. H., Raich N., Dubart A., Beaupain D., Pryor M., Kushner J., Cohen-Solal M., Goossens M. Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA. J Biol Chem. 1986 Jul 25;261(21):9825–9831. [PubMed] [Google Scholar]
  25. Rytka J., Bilinski T., Labbe-Bois R. Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda. Biochem J. 1984 Mar 1;218(2):405–413. doi: 10.1042/bj2180405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Straka J. G., Kushner J. P. Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase. Biochemistry. 1983 Sep 27;22(20):4664–4672. doi: 10.1021/bi00289a009. [DOI] [PubMed] [Google Scholar]
  28. Urban-Grimal D., Labbe-Bois R. Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis. Mol Gen Genet. 1981;183(1):85–92. doi: 10.1007/BF00270144. [DOI] [PubMed] [Google Scholar]
  29. de Verneuil H., Grandchamp B., Beaumont C., Picat C., Nordmann Y. Uroporphyrinogen decarboxylase structural mutant (Gly281----Glu) in a case of porphyria. Science. 1986 Nov 7;234(4777):732–734. doi: 10.1126/science.3775362. [DOI] [PubMed] [Google Scholar]
  30. de Verneuil H., Grandchamp B., Nordmann Y. Some kinetic properties of human red cell uroporphyrinogen decarboxylase. Biochim Biophys Acta. 1980 Jan 11;611(1):174–186. doi: 10.1016/0005-2744(80)90053-4. [DOI] [PubMed] [Google Scholar]
  31. de Verneuil H., Sassa S., Kappas A. Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. A single enzyme catalyzing the four sequential decarboxylations of uroporphyrinogens I and III. J Biol Chem. 1983 Feb 25;258(4):2454–2460. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES