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. 1993 Feb 15;290(Pt 1):279–287. doi: 10.1042/bj2900279

Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain.

P Spencer 1, P M Jordan 1
PMCID: PMC1132412  PMID: 8439296

Abstract

5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity. The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17. Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol. Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions. On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD. On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit. The formation of the first disulphide led to the loss of catalytic activity. Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands. While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity. The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134. Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E. coli.

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Selected References

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  1. Anderson P. M., Desnick R. J. Purification and properties of delta-aminolevulinate dehydrase from human erythrocytes. J Biol Chem. 1979 Aug 10;254(15):6924–6930. [PubMed] [Google Scholar]
  2. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  3. Bevan D. R., Bodlaender P., Shemin D. Mechanism of porphobilinogen synthase. Requirement of Zn2+ for enzyme activity. J Biol Chem. 1980 Mar 10;255(5):2030–2035. [PubMed] [Google Scholar]
  4. Bishop T. R., Cohen P. J., Boyer S. H., Noyes A. N., Frelin L. P. Isolation of a rat liver delta-aminolevulinate dehydrase (ALAD) cDNA clone: evidence for unequal ALAD gene dosage among inbred mouse strains. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5568–5572. doi: 10.1073/pnas.83.15.5568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boese Q. F., Spano A. J., Li J. M., Timko M. P. Aminolevulinic acid dehydratase in pea (Pisum sativum L.). Identification of an unusual metal-binding domain in the plant enzyme. J Biol Chem. 1991 Sep 15;266(26):17060–17066. [PubMed] [Google Scholar]
  6. Dent A. J., Beyersmann D., Block C., Hasnain S. S. Two different zinc sites in bovine 5-aminolevulinate dehydratase distinguished by extended X-ray absorption fine structure. Biochemistry. 1990 Aug 28;29(34):7822–7828. doi: 10.1021/bi00486a007. [DOI] [PubMed] [Google Scholar]
  7. Echelard Y., Dymetryszyn J., Drolet M., Sasarman A. Nucleotide sequence of the hemB gene of Escherichia coli K12. Mol Gen Genet. 1988 Nov;214(3):503–508. doi: 10.1007/BF00330487. [DOI] [PubMed] [Google Scholar]
  8. GIBSON K. D., NEUBERGER A., SCOTT J. J. The purification and properties of delta-aminolaevulic acid dehydrase. Biochem J. 1955 Dec;61(4):618–629. doi: 10.1042/bj0610618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gibbs P. N., Chaudhry A. G., Jordan P. M. Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes. Biochem J. 1985 Aug 15;230(1):25–34. doi: 10.1042/bj2300025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gibbs P. N., Gore M. G., Jordan P. M. Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase. Biochem J. 1985 Feb 1;225(3):573–580. doi: 10.1042/bj2250573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henderson L. E., Copeland T. D., Sowder R. C., Smythers G. W., Oroszlan S. Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses. J Biol Chem. 1981 Aug 25;256(16):8400–8406. [PubMed] [Google Scholar]
  12. Jacobson G. R., Schaffer M. H., Stark G. R., Vanaman T. C. Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J Biol Chem. 1973 Oct 10;248(19):6583–6591. [PubMed] [Google Scholar]
  13. Jaffe E. K., Salowe S. P., Chen N. T., DeHaven P. A. Porphobilinogen synthase modification with methylmethanethiosulfonate. A protocol for the investigation of metalloproteins. J Biol Chem. 1984 Apr 25;259(8):5032–5036. [PubMed] [Google Scholar]
  14. Jordan P. M., Gibbs P. N. Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes. Biochem J. 1985 May 1;227(3):1015–1020. doi: 10.1042/bj2271015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  16. Li J. M., Russell C. S., Cosloy S. D. The structure of the Escherichia coli hemB gene. Gene. 1989 Jan 30;75(1):177–184. doi: 10.1016/0378-1119(89)90394-6. [DOI] [PubMed] [Google Scholar]
  17. Li J. M., Umanoff H., Proenca R., Russell C. S., Cosloy S. D. Cloning of the Escherichia coli K-12 hemB gene. J Bacteriol. 1988 Feb;170(2):1021–1025. doi: 10.1128/jb.170.2.1021-1025.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liedgens W., Lütz C., Schneider H. A. Molecular properties of 5-aminolevulinic acid dehydratase from Spinacia oleracea. Eur J Biochem. 1983 Sep 1;135(1):75–79. doi: 10.1111/j.1432-1033.1983.tb07619.x. [DOI] [PubMed] [Google Scholar]
  19. MAUZERALL D., GRANICK S. The occurrence and determination of delta-amino-levulinic acid and porphobilinogen in urine. J Biol Chem. 1956 Mar;219(1):435–446. [PubMed] [Google Scholar]
  20. Maret W., Andersson I., Dietrich H., Schneider-Bernlöhr H., Einarsson R., Zeppezauer M. Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state. Eur J Biochem. 1979 Aug 1;98(2):501–512. doi: 10.1111/j.1432-1033.1979.tb13211.x. [DOI] [PubMed] [Google Scholar]
  21. Nandi D. L., Baker-Cohen K. F., Shemin D. Delta-aminolevulinic acid dehydratase of Rhodopseudomonas spheroides. J Biol Chem. 1968 Mar 25;243(6):1224–1230. [PubMed] [Google Scholar]
  22. Nandi D. L., Shemin D. Delta-aminolevulinic acid dehydratase of Rhodopseudomonas spheroides. 3. Mechanism of porphobilinogen synthesis. J Biol Chem. 1968 Mar 25;243(6):1236–1242. [PubMed] [Google Scholar]
  23. Nandi D. L., Shemin D. Delta-aminolevulinic acid dehydratase of Rhodopseudomonas spheroides. II. Association to polymers and dissociation to subunits. J Biol Chem. 1968 Mar 25;243(6):1231–1235. [PubMed] [Google Scholar]
  24. Spencer P., Scawen M. D., Atkinson T., Gore M. G. The identification of a structurally important cysteine residue in the glycerol dehydrogenase from Bacillus stearothermophilus. Biochim Biophys Acta. 1991 Mar 4;1073(2):386–393. doi: 10.1016/0304-4165(91)90147-9. [DOI] [PubMed] [Google Scholar]
  25. THIERS R. E. Contamination in trace element analysis and its control. Methods Biochem Anal. 1957;5:273–335. doi: 10.1002/9780470110218.ch6. [DOI] [PubMed] [Google Scholar]
  26. Tsukamoto I., Yoshinaga T., Sano S. The role of zinc with special reference to the essential thiol groups in delta-aminolevulinic acid dehydratase of bovine liver. Biochim Biophys Acta. 1979 Sep 12;570(1):167–178. doi: 10.1016/0005-2744(79)90211-0. [DOI] [PubMed] [Google Scholar]
  27. Wetmur J. G., Bishop D. F., Cantelmo C., Desnick R. J. Human delta-aminolevulinate dehydratase: nucleotide sequence of a full-length cDNA clone. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7703–7707. doi: 10.1073/pnas.83.20.7703. [DOI] [PMC free article] [PubMed] [Google Scholar]

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