Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Apr 15;371(Pt 2):351–360. doi: 10.1042/BJ20021394

Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803.

Mark Shepherd 1, James D Reid 1, C Neil Hunter 1
PMCID: PMC1223276  PMID: 12489983

Abstract

Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX (MgP) at the C(6) propionate side chain to form magnesium protoporphyrin IX monomethylester (MgPME). Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-L-methionine (SAM)-binding proteins. These searches suggest that ChlM contains a seven-stranded beta-sheet, common among small-molecule methyltransferases. Steady-state kinetic assays were performed using magnesium deuteroporphyrin IX (MgD), a more water-soluble substrate analogue of MgP. Initial rate studies showed that the reaction proceeds via a ternary complex. Product (S-adenosyl-L-homocysteine; SAH) inhibition was used to investigate the kinetic mechanism further. SAH was shown to exhibit competitive inhibition with respect to SAM, and mixed inhibition with respect to MgD. This is indicative of a random binding mechanism, whereby SAH may bind productively to either free enzyme or a ChlM-MgD complex. Our results provide an overview of the steady-state kinetics for this enzyme, which are significant given the role of MgP and MgPME in plastid-to-nucleus signalling and their likely critical role in the regulation of this biosynthetic pathway.

Full Text

The Full Text of this article is available as a PDF (405.4 KB).

Selected References

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

  1. Addlesee H. A., Hunter C. N. Physical mapping and functional assignment of the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides. J Bacteriol. 1999 Dec;181(23):7248–7255. doi: 10.1128/jb.181.23.7248-7255.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Al-Karadaghi S., Hansson M., Nikonov S., Jönsson B., Hederstedt L. Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis. Structure. 1997 Nov 15;5(11):1501–1510. doi: 10.1016/s0969-2126(97)00299-2. [DOI] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  5. Bleasby A. J., Akrigg D., Attwood T. K. OWL--a non-redundant composite protein sequence database. Nucleic Acids Res. 1994 Sep;22(17):3574–3577. [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Brocklehurst K., Crook E. M., Wharton C. W. The kinetic analysis of hydrolytic enzyme catalyses: Consequences of non-productive binding. FEBS Lett. 1968 Nov;2(1):69–73. doi: 10.1016/0014-5793(68)80103-6. [DOI] [PubMed] [Google Scholar]
  8. Cheng X., Kumar S., Posfai J., Pflugrath J. W., Roberts R. J. Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. Cell. 1993 Jul 30;74(2):299–307. doi: 10.1016/0092-8674(93)90421-l. [DOI] [PubMed] [Google Scholar]
  9. Cheng X., Roberts R. J. AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res. 2001 Sep 15;29(18):3784–3795. doi: 10.1093/nar/29.18.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu Z., Hu Y., Konishi K., Takata Y., Ogawa H., Gomi T., Fujioka M., Takusagawa F. Crystal structure of glycine N-methyltransferase from rat liver. Biochemistry. 1996 Sep 17;35(37):11985–11993. doi: 10.1021/bi961068n. [DOI] [PubMed] [Google Scholar]
  11. Gibson L. C., Hunter C. N. The bacteriochlorophyll biosynthesis gene, bchM, of Rhodobacter sphaeroides encodes S-adenosyl-L-methionine: Mg protoporphyrin IX methyltransferase. FEBS Lett. 1994 Sep 26;352(2):127–130. doi: 10.1016/0014-5793(94)00934-1. [DOI] [PubMed] [Google Scholar]
  12. Gibson L. C., Jensen P. E., Hunter C. N. Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex. Biochem J. 1999 Jan 15;337(Pt 2):243–251. [PMC free article] [PubMed] [Google Scholar]
  13. Gibson L. C., Marrison J. L., Leech R. M., Jensen P. E., Bassham D. C., Gibson M., Hunter C. N. A putative Mg chelatase subunit from Arabidopsis thaliana cv C24. Sequence and transcript analysis of the gene, import of the protein into chloroplasts, and in situ localization of the transcript and protein. Plant Physiol. 1996 May;111(1):61–71. doi: 10.1104/pp.111.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gibson L. C., Willows R. D., Kannangara C. G., von Wettstein D., Hunter C. N. Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1941–1944. doi: 10.1073/pnas.92.6.1941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gomi T., Tanihara K., Date T., Fujioka M. Rat guanidinoacetate methyltransferase: mutation of amino acids within a common sequence motif of mammalian methyltransferase does not affect catalytic activity but alters proteolytic susceptibility. Int J Biochem. 1992 Oct;24(10):1639–1649. doi: 10.1016/0020-711x(92)90182-z. [DOI] [PubMed] [Google Scholar]
  16. Gorchein A. Magnesium protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Studies with whole cells. Biochem J. 1972 Mar;127(1):97–106. doi: 10.1042/bj1270097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gribskov M., McLachlan A. D., Eisenberg D. Profile analysis: detection of distantly related proteins. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4355–4358. doi: 10.1073/pnas.84.13.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harmer S. L., Hogenesch J. B., Straume M., Chang H. S., Han B., Zhu T., Wang X., Kreps J. A., Kay S. A. Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science. 2000 Dec 15;290(5499):2110–2113. doi: 10.1126/science.290.5499.2110. [DOI] [PubMed] [Google Scholar]
  19. Henikoff S., Henikoff J. G. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915–10919. doi: 10.1073/pnas.89.22.10915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heyes D. J., Martin G. E., Reid R. J., Hunter C. N., Wilks H. M. NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation. FEBS Lett. 2000 Oct 13;483(1):47–51. doi: 10.1016/s0014-5793(00)02081-0. [DOI] [PubMed] [Google Scholar]
  21. Hinchigeri S. B., Hundle B., Richards W. R. Demonstration that the BchH protein of Rhodobacter capsulatus activates S-adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase. FEBS Lett. 1997 May 5;407(3):337–342. doi: 10.1016/s0014-5793(97)00371-2. [DOI] [PubMed] [Google Scholar]
  22. Jensen P. E., Gibson L. C., Henningsen K. W., Hunter C. N. Expression of the chlI, chlD, and chlH genes from the Cyanobacterium synechocystis PCC6803 in Escherichia coli and demonstration that the three cognate proteins are required for magnesium-protoporphyrin chelatase activity. J Biol Chem. 1996 Jul 12;271(28):16662–16667. doi: 10.1074/jbc.271.28.16662. [DOI] [PubMed] [Google Scholar]
  23. Jensen P. E., Gibson L. C., Hunter C. N. Determinants of catalytic activity with the use of purified I, D and H subunits of the magnesium protoporphyrin IX chelatase from Synechocystis PCC6803. Biochem J. 1998 Sep 1;334(Pt 2):335–344. doi: 10.1042/bj3340335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jensen P. E., Gibson L. C., Shephard F., Smith V., Hunter C. N. Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase. FEBS Lett. 1999 Jul 23;455(3):349–354. doi: 10.1016/s0014-5793(99)00909-6. [DOI] [PubMed] [Google Scholar]
  25. Jensen P. E., Willows R. D., Petersen B. L., Vothknecht U. C., Stummann B. M., Kannangara C. G., von Wettstein D., Henningsen K. W. Structural genes for Mg-chelatase subunits in barley: Xantha-f, -g and -h. Mol Gen Genet. 1996 Mar 7;250(4):383–394. doi: 10.1007/BF02174026. [DOI] [PubMed] [Google Scholar]
  26. Johanningmeier U. Possible control of transcript levels by chlorophyll precursors in Chlamydomonas. Eur J Biochem. 1988 Nov 1;177(2):417–424. doi: 10.1111/j.1432-1033.1988.tb14391.x. [DOI] [PubMed] [Google Scholar]
  27. Jones D. T. GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J Mol Biol. 1999 Apr 9;287(4):797–815. doi: 10.1006/jmbi.1999.2583. [DOI] [PubMed] [Google Scholar]
  28. Kagan R. M., Clarke S. Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch Biochem Biophys. 1994 May 1;310(2):417–427. doi: 10.1006/abbi.1994.1187. [DOI] [PubMed] [Google Scholar]
  29. Karger G. A., Reid J. D., Hunter C. N. Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex. Biochemistry. 2001 Aug 7;40(31):9291–9299. doi: 10.1021/bi010562a. [DOI] [PubMed] [Google Scholar]
  30. Keller Y., Bouvier F., d'Harlingue A., Camara B. Metabolic compartmentation of plastid prenyllipid biosynthesis--evidence for the involvement of a multifunctional geranylgeranyl reductase. Eur J Biochem. 1998 Jan 15;251(1-2):413–417. doi: 10.1046/j.1432-1327.1998.2510413.x. [DOI] [PubMed] [Google Scholar]
  31. Kropat J., Oster U., Rüdiger W., Beck C. F. Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):14168–14172. doi: 10.1073/pnas.94.25.14168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kropat J., Oster U., Rüdiger W., Beck C. F. Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus. Plant J. 2000 Nov;24(4):523–531. doi: 10.1046/j.1365-313x.2000.00898.x. [DOI] [PubMed] [Google Scholar]
  33. Lemer C. M., Rooman M. J., Wodak S. J. Protein structure prediction by threading methods: evaluation of current techniques. Proteins. 1995 Nov;23(3):337–355. doi: 10.1002/prot.340230308. [DOI] [PubMed] [Google Scholar]
  34. Mochizuki N., Brusslan J. A., Larkin R., Nagatani A., Chory J. Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):2053–2058. doi: 10.1073/pnas.98.4.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oster U., Bauer C. E., Rüdiger W. Characterization of chlorophyll a and bacteriochlorophyll a synthases by heterologous expression in Escherichia coli. J Biol Chem. 1997 Apr 11;272(15):9671–9676. doi: 10.1074/jbc.272.15.9671. [DOI] [PubMed] [Google Scholar]
  36. Russell R. B., Copley R. R., Barton G. J. Protein fold recognition by mapping predicted secondary structures. J Mol Biol. 1996 Jun 14;259(3):349–365. doi: 10.1006/jmbi.1996.0325. [DOI] [PubMed] [Google Scholar]
  37. Schluckebier G., Zhong P., Stewart K. D., Kavanaugh T. J., Abad-Zapatero C. The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism. J Mol Biol. 1999 Jun 4;289(2):277–291. doi: 10.1006/jmbi.1999.2788. [DOI] [PubMed] [Google Scholar]
  38. Schubert H. L., Raux E., Wilson K. S., Warren M. J. Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis. Biochemistry. 1999 Aug 17;38(33):10660–10669. doi: 10.1021/bi9906773. [DOI] [PubMed] [Google Scholar]
  39. Skinner M. M., Puvathingal J. M., Walter R. L., Friedman A. M. Crystal structure of protein isoaspartyl methyltransferase: a catalyst for protein repair. Structure. 2000 Nov 15;8(11):1189–1201. doi: 10.1016/s0969-2126(00)00522-0. [DOI] [PubMed] [Google Scholar]
  40. Susek R. E., Ausubel F. M., Chory J. Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development. Cell. 1993 Sep 10;74(5):787–799. doi: 10.1016/0092-8674(93)90459-4. [DOI] [PubMed] [Google Scholar]
  41. Taylor W. R. A flexible method to align large numbers of biological sequences. J Mol Evol. 1988 Dec;28(1-2):161–169. doi: 10.1007/BF02143508. [DOI] [PubMed] [Google Scholar]
  42. Woodcock S. C., Warren M. J. Evidence for a covalent intermediate in the S-adenosyl-L-methionine-dependent transmethylation reaction catalysed by sirohaem synthase. Biochem J. 1996 Jan 15;313(Pt 2):415–421. doi: 10.1042/bj3130415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yee W. C., Eglsaer S. J., Richards W. R. Confirmation of a ping-pong mechanism for S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase of etiolated wheat by an exchange reaction. Biochem Biophys Res Commun. 1989 Jul 14;162(1):483–490. doi: 10.1016/0006-291x(89)92023-8. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES