Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Structure and regulation of yeast HEM3, the gene for porphobilinogen deaminase


Porphobilinogen deaminase is the third enzyme in the heme biosynthetic pathway. hem3 mutants in Saccharomyces cerevisiae are deficient in porphobilinogen deaminase activity. We have isolated the HEM3 gene by complementation of the heme auxotrophy of a hem3 mutant. Sequence analysis reveals an open reading frame of 981 nucleotides. The derived amino acid sequence of the protein encoded by HEM3 shows extensive homology to the reported sequences for porphobilinogen deaminase from a number of other sources, indicating that HEM3 is the structural gene for porphobilinogen deaminase. Earlier reports have suggested that expression of HEM3 is induced by porphobilinogen, the substrate of the encoded enzyme. We have investigated the transcription of HEM3 and have found that it is not affected by the ability of the cell to make porphobilinogen or heme. However, we have found that HAP2 and HAP3 gene products are involved in the expression of HEM3. An important element required for expression of HEM3 has been localized to a small region that contains a sequence homologous to the HAP2-3-4 binding sites of several genes including HEM1. These findings suggest that HEM3 expression is regulated in the same manner as that of HEM1 which encodes the first enzyme of the heme biosynthetic pathway.

This is a preview of subscription content, log in to check access.


  1. Alefounder PA, Abell C, Battersby AR (1988) The sequence of hemC, hemD and two additional E. coli genes. Nucleic Acids Res 16:9871

  2. Anderson PM, Desnick RJ (1980) Purification and properties of uroporphyrinogen I synthase from human erythrocytes. J Biol Chem 255:1993–1999

  3. Bard M (1972) Biochemical and genetic aspects of nystatin resistance in Saccharomyces cerevisiae. J Bacteriol 111:649–657

  4. Bard M, Woods R, Haslen J (1974) Porphyrin mutants of Saccharomyyces cerevisiae: correlated lesions in sterol and fatty acid biosynthesis. Biochem Biophys Res Commun 56:324–330

  5. Beaumont C, Grandchamp B, Bogard M, de Veneuil H, Nordman Y (1986) Porphobilinogen deaminase is unstable in the absence of its substrate. Biochim Biophys Acta 882:384–388

  6. Beaumont C, Porcher C, Picat C, Nordmann Y, Grandchamp B (1989) The mouse porphobilinogen deaminase gene — structural organization, sequence, and transcriptional analysis. J Biol Chem 264:14829–14834

  7. Bogorad L (1963) Enzymatic mechanisms in porphyrin synthesis: possible enzymatic blocks in porphyrias. Ann NY Acad Sci 104:676–688

  8. Carlson M, Botstein D (1982) Two differentially regulated mRNAs with different 5′ ends encode secreted and intracellular forms of yeast invertase. Cell 28:145–154

  9. Dobson MJ, Tuite MF, Roberts NA, Kingsman AJ, Kingsman SM (1982) Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res 10:2625–2637

  10. Forsburg SL, Guarente L (1988) Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site. Mol Cell Biol 8:647–654

  11. Gellefors PL, Saltzgaber-Müller J, Douglas MG (1986) Selection by genetic complementation and characterization of the gene coding for the yeast porphobilinogen deaminase. Biochem J 240:673–677

  12. Gollub EG, Liu K-P, Dayan J, Adlersberg M, Sprinson DB (1977) Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene. J Biol Chem 252:2846–2854

  13. Guarente L (1983) Yeast promoters and lacZ fusions designed to study the expression of cloned genes in yeast. Methods Enzymol 101:181–191

  14. Guarente L, Mason T (1983) Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell 32:1279–1286

  15. Guarente L, Lalonde B, Gifford P, Alani E (1984) Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae. Cell 36: 503–511

  16. Higuchi M, Bogorad L (1975) The purification and properties of uroporphyrinogen I synthases and uroporphyrinogen III cosynthase. Interactions between the enzymes. Ann NY Acad Sci 244:401–418

  17. Hodge MR, Kim G, Singh K, Cumsky MG (1989) Inverse regulation of the yeast COX5 genes by oxygen and heme. Mol Cell Biol 9:1958–1964

  18. Hörtner H, Ammerer G, Hartter E, Hamilton B, Rytka J, Bilinkski T, Ruis H (1982) Regulation of synthesis of catalases and iso-1cytochrome c in Saccharomyces cerevisiae by glucose, oxygen and heme. Eur J Biochem 128:179–184

  19. Jordan PM, Shemin D (1973) Purification and properties of uroporphyrinogen I synthetase from Rhodopseudomonas spheroides. J Biol Chem 248:1019–1024

  20. Jordan PM, Warren MJ (1987) Evidence for a dipyrromethane cofactor at the catalytic site of E. coli porphobilinogen deaminase. FEBS Lett 225:87–92

  21. Jordan PM, Thomas SD, Warren MJ (1988a) Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12. Biochem J 254:427–435

  22. Jordan PM, Warren MJ, Williams HJ, Stolowich NJ, Roessner CA, Grant SK, Scott AI (1988b) Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase. FEBS Lett 235:189–193

  23. Karst F, Lacroute F (1973) Isolation of pleiotropic yeast mutants requiring ergosterol for growth. Biochem Biophys Res Commun 52:741–747

  24. Keng T, Guarente L (1987) Multiple regulatory systems result in constitutive expression of the yeast HEM1 gene. Proc Natl Acad Sci USA 84:9113–9117

  25. Kurtz MB, Marrinan J (1989) Isolation of Hem3 from Candida albicans by sequential gene disruption. Mol Gen Genet 217:47–52

  26. Lowry CV, Lieber R (1986) Negative regulation of the Saccharomyces cerevisiae ANB1 gene by heme, as mediated by the ROX1 gene product. Mol Cell Biol 6:4145–4148

  27. Marck C (1988) ‘DNA Strider’; a ‘C’ program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res 16:1829–1836

  28. Mattoon JR, Lancashire WE, Sanders HK, Carvajal E, Malamud DR, Braz RC, Panek AD (1979) Oxygen and catabolite regulation of hemoprotein biosynthesis in yeast. In: Caughey WJ (ed) Biochemical and Clinical Aspects of Oxygen. Academic Press, New York, pp 421–435

  29. Myers AM, Tzagoloff A, Kinney DM, Lusty CJ (1986) Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene 45:299–310

  30. Ng R, Abelson J (1980) Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 77:3912–3916

  31. Pfeifer K, Arcangioli B, Guarente L (1987) Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell 49:9–18

  32. Prezant T, Pfeifer K, Guarente L (1987) Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation. Mol Cell Biol 7:3252–3259

  33. Raich N, Romeo PH, Dubart A, Beaupain D, Cohen-Solal M, Goossens M (1986) Molecular cloning and complete primary sequence of human erythrocyte porphobilinogen deaminase. Nucleic Acids Res 14:5955–5968

  34. Resnick M, Mortimer R (1966) Unsaturated fatty acid mutants of Saccharomyces cerevisiae. J Bacteriol 92:597–600

  35. Rothstein R (1983) One step gene disruption in yeast. Methods Enzymol 101:202–211

  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  37. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

  38. Scott AI, Roessner CA, Stolowich NJ, Karuso P, Williams HJ, Grant SK, Gonzalez MD, Hoshino T (1988) Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase. Biochemistry 7:7984–7990

  39. Scott AI, Clemens KR, Stolowich NJ, Santander PJ, Gonzalez MD, Roessner CA (1989) Reconstitution of apo-porphobilinogen deaminase: structural changes induced by cofactor binding. FEBS Letts 242:319–324

  40. Sharif AL, Smith AG, Abell C (1989) Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis. Eur J Biochem 184:353–359

  41. Sharp PM, Li W-H (1987) The codon adaptation index — a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15:1281–1295

  42. Sharp PM, Tuohy TM, Mosunski KR (1986) Codon usage in yeast: cluster analysis clearly differentiates high and lowly expressed genes. Nucleic Acids Res 13:5125–5143

  43. Sherman F, Fink GR, Hicks JB (1986) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  44. Stubnicer A-C, Picat C, Grandchamp B (1988) Rat porphobilinogen deaminase cDNA: nucleotide sequence of the erythropoietic form. Nucleic Acids Res 16:3102

  45. Thomas SD, Jordan PM (1986) Nucleotide sequence of the hemC locus encoding porphobilinogen deaminase of Escherichia coli K12. Nucleic Acids Res 14:6215–6226

  46. Thorsness M, Schafer W, D'Ari L, Rine J (1989) Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae. Mol Cell Biol 9:5702–5712

  47. Trawick JD, Wright RM, Poyton RO (1989a) Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene. J Biol Chem 264:7005–7008

  48. Trawick JD, Rogness C, Poyton RO (1989b) Identification of an upstream activation sequence and other cis-acting elements required for transcription of COX6 from Saccharomyces cerevisiae. Mol Cell Biol 9:5350–5358

  49. Urban-Grimal D, Labbe-Bois R (1981) Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis. Mol Gen Genet 183:85–92

  50. Williams DC, Morgan GS, McDonald E, Battersby AR (1981) Purification of porphobilinogen deaminase from E. gracilis and studies of its kinetics. Biochem J 193:301–310

Download references

Author information

Correspondence to Teresa Keng.

Additional information

Communicated by D.Y. Thomas

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Keng, T., Richard, C. & Larocque, R. Structure and regulation of yeast HEM3, the gene for porphobilinogen deaminase. Molec. Gen. Genet. 234, 233–243 (1992).

Download citation

Key words

  • Yeast
  • Heme
  • Sequence
  • Regulation