Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.
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.
Mialoundama
Alexis Samba
AS
Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 67084 Strasbourg cedex, France.
Heintz
Dimitri
D
Jadid
Nurul
N
Nkeng
Paul
P
Rahier
Alain
A
Deli
Jozsef
J
Camara
Bilal
B
Bouvier
Florence
F
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*
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
Plant J. 1994 Jul;6(1):45-54
7920703
Eur J Biochem. 2000 Sep;267(17):5509-19
10951210
FEBS Lett. 2002 Mar 27;515(1-3):133-6
11943208
Arch Biochem Biophys. 1997 Oct 1;346(1):53-64
9328284
Nature. 1974 Jul 19;250(463):194-9
4368490
Chem Biol. 2004 Jan;11(1):121-6
15113001
Eur J Biochem. 2000 Nov;267(21):6346-52
11029576
Biochemistry. 2007 May 8;46(18):5437-45
17428035
Annu Rev Microbiol. 1997;51:629-59
9343362
Biochem J. 1953 Jun;54(3):437-42
13058921
Mol Gen Genet. 2000 Apr;263(3):423-32
10821176
Mol Cell Proteomics. 2009 Sep;8(9):2159-69
19497847
Angew Chem Int Ed Engl. 2000 Aug 18;39(16):2812-2833
11027983
Nat Prod Rep. 2002 Dec;19(6):761-72
12521268
Science. 2010 Apr 30;328(5978):624-7
20431015
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1687-92
11172012
J Bacteriol. 1990 Dec;172(12):6704-12
2254247
Eur J Biochem. 1988 Dec 1;178(1):197-207
3203689
Biochem J. 1969 Aug;114(1):71-3
5810070
Biochem J. 1996 May 1;315 ( Pt 3):869-74
8645170
J Am Chem Soc. 2008 Apr 9;130(14):4906-13
18345677
J Biol Chem. 2009 Apr 3;284(14):9160-7
19158086
Mol Gen Genet. 1999 Oct;262(3):453-61
10589832
Biochem Biophys Res Commun. 2004 Sep 24;322(3):905-10
15336549
FEMS Microbiol Lett. 2000 Jun 1;187(1):95-101
10828407
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2905-10
11226339
Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):11102-7
10995464
Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11784-9
17606904
Protein Sci. 2001 Sep;10(9):1712-28
11514662
Plant Cell. 1994 Aug;6(8):1107-21
7919981
Anal Biochem. 1985 Oct;150(1):76-85
3843705
J Biol Chem. 2010 Apr 16;285(16):12109-20
20178989
Anal Biochem. 2004 Jan 1;324(1):131-6
14654055
Plant J. 1995 Sep;8(3):417-24
7550379