Identification and characterization of a unique cysteine residue proximal to the catalytic site of Arabidopsis thaliana carotenoid cleavage enzyme 1.
AtCCD1 and AtNCED3 are related carotenoid cleavage enzymes from Arabidopsis thaliana that catalyze the oxidative cleavage of, respectively, the 9,10 (9',10') double bonds of carotenoid substrates such as beta-carotene, and the 11,12 double bond of 9-cis epoxycarotenoids. Although the cellular and cleavage functionalities of these enzymes have been reported, their mechanisms and related structural environments mediating these disparate specificities in homologous enzymes have not been well characterized. By relating the differences observed in UV and visible light absorption and Cu(II) electron paramagnetic signals to variations in sequence alignments and 3-D homology models of the two A. thaliana enzymes, we identified a putatively proximal cysteine residue (Cys352) in AtCCD1 that is not conserved in AtNCED3. Spectral analysis of the Cys to Ala mutant confirmed its uniqueness and proximity to the metal binding site, but precluded any role for the residue in the mediation of the observed metal binding affinity or associated steric constraint differences. Further analysis of kinetic substrate cleavage properties indicated a decrease in Vmax and a subtle increase in Km for the C352A mutant compared with those observed for the wild-type, thus confirming catalytic site proximity and suggesting possible roles for the unique cysteine in the modulation of substrate affinity and (or) the reaction rate of AtCCD1.
Plant Biotechnology Institute, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, Canada.
Shukui
S
Guo
Jason
J
Boyd
Ramaswami
R
Sammynaiken
Michèle C
MC
Loewen
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D029681:Arabidopsis Proteins; D008670:Metals; D010940:Plant Proteins; D011994:Recombinant Proteins; D010105:Oxygenases; D049308:Dioxygenases; C431939:carotenoid cleavage dioxygenase 1; C109043:9-cis-epoxy-carotenoid dioxygenase; D003545:Cysteine