Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1996 Sep;112(1):105–114. doi: 10.1104/pp.112.1.105

The Chlorophyll Biosynthetic Enzyme Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase (Characterization and Partial Purification from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803).

D W Bollivar 1, S I Beale 1
PMCID: PMC157929  PMID: 12226378

Abstract

A universal structural feature of chlorophyll molecules is the isocyclic ring. This ring is formed by the action of the enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, which catalyzes a complex reaction in which Mg-protoporphyrin IX monomethyl ester is converted to divinyl protochlorophyllide (also called Mg-2,4-divinylpheoporphyrin a5), with the participation of NADPH and O2. Cyclase activity was demonstrated in lysed Chlamydomonas reinhardtii chloroplasts and extracts of Synechocystis sp. PCC 6803. The yield of the reaction product was increased by the addition of catalase and ascorbate or isoascorbate to the incubation mixture. These compounds may act by preventing degradation of the tetrapyrroles by reactive oxygen species. Cyclase activity from C. reinhardtii was not inhibited by the flavoprotein inhibitor quinacrine or by the hemoprotein inhibitors CO, KCN, or NaN3. In contrast, cyclase activity in extracts of C. reinhardtii and Synechocystis sp. PCC 6803 was inhibited by chelators of Fe, suggesting that nonheme Fe is involved in the reaction. Cyclase in lysed C. reinhardtii chloroplasts was associated with the membranes, and attempts to further fractionate or solubilize the activity were unsuccessful. In contrast, cyclase in Synechocystis sp. PCC 6803 extracts could be separated into soluble and membrane components, both of which were required for reconstitution of activity. The membrane component retained activity after it was solubilized by the detergent n-octyl-[beta]-D-glucopyranoside in the presence of glycerol and Mg2+. The solubilized membrane component was purified further by dye-affinity and ion-exchange chromatography.

Full Text

The Full Text of this article is available as a PDF (1,023.3 KB).

Selected References

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

  1. Bednarik D. P., Hoober J. K. Biosynthesis of a chlorophyllide b-like pigment in phenanthroline-treated Chlamydomonas reinhardtii y-1. Arch Biochem Biophys. 1985 Jul;240(1):369–379. doi: 10.1016/0003-9861(85)90042-6. [DOI] [PubMed] [Google Scholar]
  2. Bollivar D. W., Jiang Z. Y., Bauer C. E., Beale S. I. Heterologous expression of the bchM gene product from Rhodobacter capsulatus and demonstration that it encodes S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase. J Bacteriol. 1994 Sep;176(17):5290–5296. doi: 10.1128/jb.176.17.5290-5296.1994. [DOI] [PMC free article] [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Fuesler T. P., Wong Y. S., Castelfranco P. A. Localization of Mg-Chelatase and Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Activities within Isolated, Developing Cucumber Chloroplasts. Plant Physiol. 1984 Jul;75(3):662–664. doi: 10.1104/pp.75.3.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kahn A., Lagneau J., Boivin P., Hakim J. Importance et rôle des groupes imidazole et sulfhydrile dans l'activité catalytique de la glucose 6 phosphate déshydrogénase érythrocytaire humaine. Biochimie. 1972;54(8):997–1004. doi: 10.1016/s0300-9084(72)80050-6. [DOI] [PubMed] [Google Scholar]
  11. Narasimhan C., Miziorko H. M. Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements. Biochemistry. 1992 Nov 17;31(45):11224–11230. doi: 10.1021/bi00160a037. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Porra R. J., Schäfer W., Katheder I., Scheer H. The derivation of the oxygen atoms of the 13(1)-oxo and 3-acetyl groups of bacteriochlorophyll a from water in Rhodobacter sphaeroides cells adapting from respiratory to photosynthetic conditions: evidence for an anaerobic pathway for the formation of isocyclic ring E. FEBS Lett. 1995 Aug 28;371(1):21–24. doi: 10.1016/0014-5793(95)00854-3. [DOI] [PubMed] [Google Scholar]
  14. Spiller S. C., Castelfranco A. M., Castelfranco P. A. Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS. Plant Physiol. 1982 Jan;69(1):107–111. doi: 10.1104/pp.69.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tripathy B. C., Rebeiz C. A. Chloroplast biogenesis: quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyll(ides) by spectrofluorometry. Anal Biochem. 1985 Aug 15;149(1):43–61. doi: 10.1016/0003-2697(85)90475-0. [DOI] [PubMed] [Google Scholar]
  16. Walker C. J., Castelfranco P. A., Whyte B. J. Synthesis of divinyl protochlorophyllide. Enzymological properties of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system. Biochem J. 1991 Jun 15;276(Pt 3):691–697. doi: 10.1042/bj2760691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES