Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina

Abstract

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1'-(HO)(2)-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K (m) and V (max) values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h(-1) mg(-1) for RgCrtC and 9.5 μM and 0.15 nmol h(-1) mg(-1) for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene.

Fig. 1

SDS-PAGE (10%) analysis of expression and IMAC purification for RgCrtC (lanes 1–3) and TrCrtC (lanes 4–6). M Precision plus protein standard; a whole cells before induction; b whole cells after induction with 0.1 mM IPTG and expression overnight at 25°C; c purified CrtCs

Fig. 2

Reaction catalyzed by Rubrivivax gelatinosus and Thiocapsa roseopersicina carotenoid 1,2-hydratase; the conversion of lycopene into 1-HO-lycopene and 1,1′-(HO)₂-lycopene (a). HPLC separation of carotenoids formed in vitro by Escherichia coli extract expressing the RgCrtC (solid line) and TrCrtC (dashed line). Peak 1, 1,1′-(HO)₂-lycopene; peak 2, 1-HO-lycopene; peak 3, lycopene (b)

Fig. 3

Michaelis–Menten plot of recombinant RgCrtC (filled circle) and TrCrtC (empty circle). The cell-free extracts were assayed with various lycopene concentrations (0.5–40 μM) in 50 mM Na₂HPO₄ sodium phosphate (pH 8.0) at 28°C for 4 h. The rates of product formation (1-HO-lycopene plus 1,1′-(HO)₂-lycopene) are plotted against varying substrate concentrations. Kinetic constants are listed in Table 1

Fig. 4

Effect of pH (a) and temperature (b) on activity of RgCrtC (filled circle) and TrCrtC (empty circle). For pH effect, measurements were performed with lycopene under standard assay conditions using different buffers: 100 mM acetate (pH 4.0, 5.0 and 6.0), 100 mM potassium phosphate (pH 6.0, 7.0, and 8.0), and 50 mM Tris–HCl (pH 8.0, 8.6 and 9.0). For temperature effect, activity assays were performed with lycopene at various temperatures (1–50°C) under standard assay conditions

Fig. 5

Enzyme stability of recombinant TrCrtC by CD spectroscopy. The purified CrtC was diluted to 0.03 mg/ml with 10 mM sodium phosphate, pH 8.0, and incubated for 5 min from 5°C to 90°C. CD assay was performed by wavelength scan from 190 to 250 nm