title: Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.
abstract: The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 67084 Strasbourg cedex, France.
authors.forename: Alexis Samba
authors.initials: AS
authors.lastname: Mialoundama
authors.forename: Dimitri
authors.initials: D
authors.lastname: Heintz
authors.forename: Nurul
authors.initials: N
authors.lastname: Jadid
authors.forename: Paul
authors.initials: P
authors.lastname: Nkeng
authors.forename: Alain
authors.initials: A
authors.lastname: Rahier
authors.forename: Jozsef
authors.initials: J
authors.lastname: Deli
authors.forename: Bilal
authors.initials: B
authors.lastname: Camara
authors.forename: Florence
authors.initials: F
authors.lastname: Bouvier
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002212:Capsicum / Q000201:enzymology*; D002338:Carotenoids / Q000096:biosynthesis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism*; D019753:Intramolecular Lyases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005420:Flavoproteins; D050505:Mutant Proteins; D010940:Plant Proteins; D005182:Flavin-Adenine Dinucleotide; D002338:Carotenoids; D009249:NADP; D010088:Oxidoreductases; D019753:Intramolecular Lyases; C070925:lycopene cyclase-isomerase; C086535:CCS protein, Capsicum annuum
references.citation: Plant J. 1994 Jul;6(1):45-54
references.pmid: 7920703
references.citation: Eur J Biochem. 2000 Sep;267(17):5509-19
references.pmid: 10951210
references.citation: FEBS Lett. 2002 Mar 27;515(1-3):133-6
references.pmid: 11943208
references.citation: Arch Biochem Biophys. 1997 Oct 1;346(1):53-64
references.pmid: 9328284
references.citation: Nature. 1974 Jul 19;250(463):194-9
references.pmid: 4368490
references.citation: Chem Biol. 2004 Jan;11(1):121-6
references.pmid: 15113001
references.citation: Eur J Biochem. 2000 Nov;267(21):6346-52
references.pmid: 11029576
references.citation: Biochemistry. 2007 May 8;46(18):5437-45
references.pmid: 17428035
references.citation: Annu Rev Microbiol. 1997;51:629-59
references.pmid: 9343362
references.citation: Biochem J. 1953 Jun;54(3):437-42
references.pmid: 13058921
references.citation: Mol Gen Genet. 2000 Apr;263(3):423-32
references.pmid: 10821176
references.citation: Mol Cell Proteomics. 2009 Sep;8(9):2159-69
references.pmid: 19497847
references.citation: Angew Chem Int Ed Engl. 2000 Aug 18;39(16):2812-2833
references.pmid: 11027983
references.citation: Nat Prod Rep. 2002 Dec;19(6):761-72
references.pmid: 12521268
references.citation: Science. 2010 Apr 30;328(5978):624-7
references.pmid: 20431015
references.citation: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1687-92
references.pmid: 11172012
references.citation: J Bacteriol. 1990 Dec;172(12):6704-12
references.pmid: 2254247
references.citation: Eur J Biochem. 1988 Dec 1;178(1):197-207
references.pmid: 3203689
references.citation: Biochem J. 1969 Aug;114(1):71-3
references.pmid: 5810070
references.citation: Biochem J. 1996 May 1;315 ( Pt 3):869-74
references.pmid: 8645170
references.citation: J Am Chem Soc. 2008 Apr 9;130(14):4906-13
references.pmid: 18345677
references.citation: J Biol Chem. 2009 Apr 3;284(14):9160-7
references.pmid: 19158086
references.citation: Mol Gen Genet. 1999 Oct;262(3):453-61
references.pmid: 10589832
references.citation: Biochem Biophys Res Commun. 2004 Sep 24;322(3):905-10
references.pmid: 15336549
references.citation: FEMS Microbiol Lett. 2000 Jun 1;187(1):95-101
references.pmid: 10828407
references.citation: Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2905-10
references.pmid: 11226339
references.citation: Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):11102-7
references.pmid: 10995464
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11784-9
references.pmid: 17606904
references.citation: Protein Sci. 2001 Sep;10(9):1712-28
references.pmid: 11514662
references.citation: Plant Cell. 1994 Aug;6(8):1107-21
references.pmid: 7919981
references.citation: Anal Biochem. 1985 Oct;150(1):76-85
references.pmid: 3843705
references.citation: J Biol Chem. 2010 Apr 16;285(16):12109-20
references.pmid: 20178989
references.citation: Anal Biochem. 2004 Jan 1;324(1):131-6
references.pmid: 14654055
references.citation: Plant J. 1995 Sep;8(3):417-24
references.pmid: 7550379