title: Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium Synechocystis sp. PCC 6803.
abstract: Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His(6)-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, k(cat), compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.
authors: Deptartment of Chemistry and Umeå Plant Science Centre, Umeå University, Umeå, Sweden.
authors: Patrik
authors: P
authors: Storm
authors: Tania
authors: T
authors: Tibiletti
authors: Michael
authors: M
authors: Hall
authors: Christiane
authors: C
authors: Funk
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D061146:Chlorophyll Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010860:Pigments, Biological / Q000737:chemistry / Q000378:metabolism; D018087:Plastids / Q000201:enzymology* / Q000235:genetics; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D061146:Chlorophyll Binding Proteins; D003067:Coenzymes; D045342:Light-Harvesting Protein Complexes; D010860:Pigments, Biological; D011994:Recombinant Proteins; D006418:Heme; D005294:Ferrochelatase
references: Cell Mol Life Sci. 2000 Dec;57(13-14):1909-26
references: 11215517
references: J Biol Chem. 1997 Oct 31;272(44):27565-71
references: 9346891
references: J Biol Chem. 2002 Feb 15;277(7):4731-7
references: 11675381
references: Photochem Photobiol Sci. 2008 Oct;7(10):1243-52
references: 18846290
references: J Biol Chem. 2012 Jan 2;287(1):682-692
references: 22090028
references: Planta. 2003 Aug;217(4):602-9
references: 12905021
references: J Biol Inorg Chem. 2006 Apr;11(3):325-33
references: 16453119
references: Plant Physiol. 2004 Aug;135(4):2379-91
references: 15326282
references: Plant J. 1998 Aug;15(4):531-41
references: 9753778
references: Plant Physiol. 2011 Apr;155(4):1735-47
references: 21081693
references: Plant Physiol. 1975 Mar;55(3):485-90
references: 16659107
references: Biochem Soc Trans. 2005 Aug;33(Pt 4):737-42
references: 16042589
references: Protein Eng Des Sel. 2008 Aug;21(8):529-36
references: 18502789
references: Annu Rev Plant Biol. 2007;58:321-46
references: 17227226
references: Biochem J. 2002 Mar 1;362(Pt 2):423-32
references: 11853551
references: Anal Biochem. 2003 May 15;316(2):223-31
references: 12711344
references: J Biol Chem. 2008 Aug 29;283(35):23685-91
references: 18593702
references: J Biol Chem. 1993 Apr 25;268(12):8541-6
references: 8473299
references: J Biol Chem. 2005 Sep 9;280(36):31595-602
references: 16027152
references: Biochemistry. 2007 Jan 9;46(1):87-94
references: 17198378
references: Protein Expr Purif. 2003 Mar;28(1):1-8
references: 12651100
references: J Bacteriol. 2008 Mar;190(6):2086-95
references: 18192382
references: Biochem Soc Trans. 2002 Aug;30(4):590-5
references: 12196143
references: Biochim Biophys Acta. 2011 Sep;1807(9):1143-51
references: 21605542
references: Physiol Plant. 2002 May;115(1):9-24
references: 12010463
references: J Phys Chem B. 2007 Nov 1;111(43):12427-38
references: 17924686
references: Trends Plant Sci. 1999 Jun;4(6):236-240
references: 10366881
references: Plant Physiol. 2005 Dec;139(4):1946-58
references: 16306143
references: Photosynth Res. 2008 Oct-Dec;98(1-3):479-88
references: 18836846
references: J Biol Chem. 2004 May 7;279(19):19977-86
references: 14981080
references: Plant Mol Biol. 2002 Nov;50(4-5):773-88
references: 12374307
references: Plant Mol Biol. 2002 May;49(2):149-60
references: 11999371
references: Anal Biochem. 2003 Jul 1;318(1):18-24
references: 12782026
references: J Biol Chem. 2004 Jul 2;279(27):27971-9
references: 15107425
references: Biochemistry. 1999 Jul 20;38(29):9397-404
references: 10413515
references: J Bacteriol. 2003 Mar;185(5):1719-25
references: 12591891
references: Mol Med. 2003 May-Aug;9(5-8):175-82
references: 14571325
references: FEBS Lett. 2001 Oct 12;506(3):291-5
references: 11602264
references: J Biol Chem. 1988 Aug 25;263(24):11675-82
references: 3042776