Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Jan;5(1):98–105. doi: 10.1002/pro.5560050112

Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.

T A Dailey 1, H A Dailey 1
PMCID: PMC2143237  PMID: 8771201

Abstract

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.

Full Text

The Full Text of this article is available as a PDF (4.0 MB).

Selected References

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

  1. Brenner D. A., Bloomer J. R. A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue. Clin Chim Acta. 1980 Jan 31;100(3):259–266. doi: 10.1016/0009-8981(80)90275-2. [DOI] [PubMed] [Google Scholar]
  2. Brenner D. A., Bloomer J. R. The enzymatic defect in variegate prophyria. Studies with human cultured skin fibroblasts. N Engl J Med. 1980 Apr 3;302(14):765–769. doi: 10.1056/NEJM198004033021401. [DOI] [PubMed] [Google Scholar]
  3. Camadro J. M., Thome F., Brouillet N., Labbe P. Purification and properties of protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. Mitochondrial location and evidence for a precursor form of the protein. J Biol Chem. 1994 Dec 23;269(51):32085–32091. [PubMed] [Google Scholar]
  4. Caughman S. W., Hentze M. W., Rouault T. A., Harford J. B., Klausner R. D. The iron-responsive element is the single element responsible for iron-dependent translational regulation of ferritin biosynthesis. Evidence for function as the binding site for a translational repressor. J Biol Chem. 1988 Dec 15;263(35):19048–19052. [PubMed] [Google Scholar]
  5. Dailey H. A., Karr S. W. Purification and characterization of murine protoporphyrinogen oxidase. Biochemistry. 1987 May 19;26(10):2697–2701. doi: 10.1021/bi00384a007. [DOI] [PubMed] [Google Scholar]
  6. Dailey T. A., Meissner P., Dailey H. A. Expression of a cloned protoporphyrinogen oxidase. J Biol Chem. 1994 Jan 14;269(2):813–815. [PubMed] [Google Scholar]
  7. Deybach J. C., de Verneuil H., Nordmann Y. The inherited enzymatic defect in porphyria variegata. Hum Genet. 1981;58(4):425–428. doi: 10.1007/BF00282829. [DOI] [PubMed] [Google Scholar]
  8. Ferreira G. C., Andrew T. L., Karr S. W., Dailey H. A. Organization of the terminal two enzymes of the heme biosynthetic pathway. Orientation of protoporphyrinogen oxidase and evidence for a membrane complex. J Biol Chem. 1988 Mar 15;263(8):3835–3839. [PubMed] [Google Scholar]
  9. Ferreira G. C., Dailey H. A. Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin. Biochem J. 1988 Mar 1;250(2):597–603. doi: 10.1042/bj2500597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hansson M., Hederstedt L. Bacillus subtilis HemY is a peripheral membrane protein essential for protoheme IX synthesis which can oxidize coproporphyrinogen III and protoporphyrinogen IX. J Bacteriol. 1994 Oct;176(19):5962–5970. doi: 10.1128/jb.176.19.5962-5970.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hansson M., Hederstedt L. Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes. J Bacteriol. 1992 Dec;174(24):8081–8093. doi: 10.1128/jb.174.24.8081-8093.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs J. M., Jacobs N. J., Sherman T. D., Duke S. O. Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley. Plant Physiol. 1991 Sep;97(1):197–203. doi: 10.1104/pp.97.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jacobs J. M., Sinclair P. R., Gorman N., Jacobs N. J., Sinclair J. F., Bement W. J., Walton H. Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes. J Biochem Toxicol. 1992 Summer;7(2):87–95. doi: 10.1002/jbt.2570070206. [DOI] [PubMed] [Google Scholar]
  14. Jacobs N. J., Jacobs J. M. Protoporphyrinogen oxidation in Rhodopseudomonas spheroides, a step in heme and bacteriochlorophyll synthesis. Arch Biochem Biophys. 1981 Oct 1;211(1):305–311. doi: 10.1016/0003-9861(81)90458-6. [DOI] [PubMed] [Google Scholar]
  15. Klemm D. J., Barton L. L. Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas. J Bacteriol. 1987 Nov;169(11):5209–5215. doi: 10.1128/jb.169.11.5209-5215.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matringe M., Camadro J. M., Labbe P., Scalla R. Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides. Biochem J. 1989 May 15;260(1):231–235. doi: 10.1042/bj2600231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Milpetz F., Argos P., Persson B. TMAP: a new email and WWW service for membrane-protein structural predictions. Trends Biochem Sci. 1995 May;20(5):204–205. doi: 10.1016/s0968-0004(00)89009-x. [DOI] [PubMed] [Google Scholar]
  18. Nishimura K., Taketani S., Inokuchi H. Cloning of a human cDNA for protoporphyrinogen oxidase by complementation in vivo of a hemG mutant of Escherichia coli. J Biol Chem. 1995 Apr 7;270(14):8076–8080. doi: 10.1074/jbc.270.14.8076. [DOI] [PubMed] [Google Scholar]
  19. Porra R. J., Falk J. E. The enzymic conversion of coproporphyrinogen 3 into protoporphyrin 9. Biochem J. 1964 Jan;90(1):69–75. doi: 10.1042/bj0900069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Poulson R., Polglase W. J. The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae. J Biol Chem. 1975 Feb 25;250(4):1269–1274. [PubMed] [Google Scholar]
  21. Sasarman A., Letowski J., Czaika G., Ramirez V., Nead M. A., Jacobs J. M., Morais R. Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12. Can J Microbiol. 1993 Dec;39(12):1155–1161. doi: 10.1139/m93-174. [DOI] [PubMed] [Google Scholar]
  22. Siepker L. J., Ford M., de Kock R., Kramer S. Purification of bovine protoporphyrinogen oxidase: immunological cross-reactivity and structural relationship to ferrochelatase. Biochim Biophys Acta. 1987 Jul 7;913(3):349–358. doi: 10.1016/0167-4838(87)90146-4. [DOI] [PubMed] [Google Scholar]
  23. Sugino H., Ishibashi K., Sakaue M., Yamashita M., Murooka Y. Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from Klebsiella aerogenes. Appl Microbiol Biotechnol. 1991 Aug;35(5):606–610. doi: 10.1007/BF00169624. [DOI] [PubMed] [Google Scholar]
  24. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES