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. 1991 Jun 15;276(Pt 3):691–697. doi: 10.1042/bj2760691

Synthesis of divinyl protochlorophyllide. Enzymological properties of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system.

C J Walker 1, P A Castelfranco 1, B J Whyte 1
PMCID: PMC1151060  PMID: 1905926

Abstract

The resolution and reconstitution of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system into a supernatant and a pellet fraction was accomplished by a procedure involving salt treatment followed by osmotic shock. Recombination of pellet and supernatant fractions was required for cyclase activity. This restoration effect could be demonstrated using either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester as the cyclase substrate in the presence or absence of S-adenosylmethionine. Pretreatment of the pellet fraction with either 8-hydroxyquinoline or desferal mesylate inhibited cyclase activity, indicating that there is a heavy-metal-ion requirement in this fraction. The cyclase supernatant protein(s) was not internalized by Sephadex G-50 and did not bind to Blue Sepharose, suggesting that it has a molecular mass of over 30 kDa and that it does not bind the cofactor NADPH. The cyclase supernatant protein did bind to MgProtoMe2-bound Sepharose and could be eluted by raising the pH to 9.7 in the presence of 4 mM-n-octyl glucoside. The pH optimum of the cyclase was 9.0. About a 40-fold purification of the cyclase supernatant protein was achieved by a combination of (NH4)2SO4 fractionation and phenyl-Sepharose chromatography.

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Selected References

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

  1. Bauer C. E., Marrs B. L. Rhodobacter capsulatus puf operon encodes a regulatory protein (PufQ) for bacteriochlorophyll biosynthesis. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7074–7078. doi: 10.1073/pnas.85.19.7074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer C. E., Young D. A., Marrs B. L. Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene. J Biol Chem. 1988 Apr 5;263(10):4820–4827. [PubMed] [Google Scholar]
  3. 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]
  4. Castelfranco P. A., Weinstein J. D., Schwarcz S., Pardo A. D., Wezelman B. E. The Mg insertion step in chlorophyll biosynthesis. Arch Biochem Biophys. 1979 Feb;192(2):592–598. doi: 10.1016/0003-9861(79)90130-9. [DOI] [PubMed] [Google Scholar]
  5. Chereskin B. M., Castelfranco P. A., Dallas J. L., Straub K. M. Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts. Arch Biochem Biophys. 1983 Oct 1;226(1):10–18. doi: 10.1016/0003-9861(83)90266-7. [DOI] [PubMed] [Google Scholar]
  6. Chereskin B. M., Castelfranco P. A. Effects of Iron and Oxygen on Chlorophyll Biosynthesis : II. OBSERVATIONS ON THE BIOSYNTHETIC PATHWAY IN ISOLATED ETIOCHLOROPLASTS. Plant Physiol. 1982 Jan;69(1):112–116. doi: 10.1104/pp.69.1.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chereskin B. M., Wong Y. S., Castelfranco P. A. In Vitro Synthesis of the Chlorophyll Isocyclic Ring : Transformation of Magnesium-Protoporphyrin IX and Magnesium-Protoporphyrin IX Monomethyl Ester into Magnesium-2,4-Divinyl Pheoporphyrin A(5). Plant Physiol. 1982 Oct;70(4):987–993. doi: 10.1104/pp.70.4.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuesler T. P., Hanamoto C. M., Castelfranco P. A. Separation of Mg-Protoporphyrin IX and Mg-Protoporphyrin IX Monomethyl Ester Synthesized de novo by Developing Cucumber Etioplasts. Plant Physiol. 1982 Feb;69(2):421–423. doi: 10.1104/pp.69.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fufsler T. P., Castelfranco P. A., Wong Y. S. Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts. Plant Physiol. 1984 Apr;74(4):928–933. doi: 10.1104/pp.74.4.928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. GRANICK S. The structural and functional relationships between heme and chlorophyll. Harvey Lect. 1948 1949;Series 44:220–245. [PubMed] [Google Scholar]
  11. Griffiths W. T. Reconstitution of chlorophyllide formation by isolated etioplast membranes. Biochem J. 1978 Sep 15;174(3):681–692. doi: 10.1042/bj1740681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hardy S. I., Castelfranco P. A., Rebeiz C. A. Effect of the hypocotyl hook on greening in etiolated cucumber cotyledons. Plant Physiol. 1970 Nov;46(5):705–707. doi: 10.1104/pp.46.5.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heyns W., De Moor P. A 3(17)beta-hydroxysteroid dehydrogenase in raterythrocytes. Conversion of 5alpha-dihydrotestosterone into 5alpha-androstane-3beta,17beta-diol and purification of the enzyme by affinity chromatography. Biochim Biophys Acta. 1974 Jul 17;358(1):1–13. doi: 10.1016/0005-2744(74)90251-4. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Nasrulhaq-Boyce A., Griffiths W. T., Jones O. T. The use of continuous assays to characterize the oxidative cyclase that synthesizes the chlorophyll isocyclic ring. Biochem J. 1987 Apr 1;243(1):23–29. doi: 10.1042/bj2430023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Walker C. J., Mansfield K. E., Rezzano I. N., Hanamoto C. M., Smith K. M., Castelfranco P. A. The magnesium-protoporphyrin IX (oxidative) cyclase system. Studies on the mechanism and specificity of the reaction sequence. Biochem J. 1988 Oct 15;255(2):685–692. [PMC free article] [PubMed] [Google Scholar]
  17. Walker C. J., Mansfield K. E., Smith K. M., Castelfranco P. A. Incorporation of atmospheric oxygen into the carbonyl functionality of the protochlorophyllide isocyclic ring. Biochem J. 1989 Jan 15;257(2):599–602. doi: 10.1042/bj2570599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wong Y. S., Castelfranco P. A., Goff D. A., Smith K. M. Intermediates in the formation of the chlorophyll isocyclic ring. Plant Physiol. 1985 Nov;79(3):725–729. doi: 10.1104/pp.79.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wong Y. S., Castelfranco P. A. Properties of the Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase System. Plant Physiol. 1985 Nov;79(3):730–733. doi: 10.1104/pp.79.3.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wong Y. S., Castelfranco P. A. Resolution and Reconstitution of Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase, the Enzyme System Responsible for the Formation of the Chlorophyll Isocyclic Ring. Plant Physiol. 1984 Jul;75(3):658–661. doi: 10.1104/pp.75.3.658. [DOI] [PMC free article] [PubMed] [Google Scholar]

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