Demonstration and Solubilization of Lycopene Cyclase from Capsicum Chromoplast Membranes

Abstract

The conversion of phytoene into fl-carotene was demonstrated previously in chromoplast membranes prepared from Capsicum fruits (B. Camara et al. 1982 Eur J Biochem 127: 255-258). The direct cyclization of lycopene into fl-carotene and the successful solubilization of the enzymic activity involved in this reaction is reported.incubation time, the reaction was stopped by the addition of 4 ml of chloroform:methanol (2:1, v/v). Carrier amounts (100 ,g) of i-carotene, y-carotene, a-carotene, and lycopene were added to facilitate the location of the reaction products. Preliminary chromatography was achieved on Silica Gel G plates developed with 40 to 60°C petroleum ether ether (99:1, v/v), which separates the bicyclic carotene fraction (a-carotene and ,Bcarotene with a higher RF value) from lycopene (lower RF value). The Through the use of isolated chromoplast membranes, we have shown that labeled phytoene is desaturated, isomerized, and cyclized to yield ,B-carotene (6). Lycopene plays a mandatory role in this sequence of reactions in that it is the direct substrate for cyclization, as shown by Camara and Moneger (7). We report here the direct cyclization oflycopene by Capsicum chromoplast membranes and the solubilization of the enzymic activity involved.MATERlALS AND METHODS Capsicum Chromoplast Membranes. The chromoplast membranes were prepared as described previously (5). The medium used for osmotic shock contained: 1 mM MgCl2, 10 mm DTT, and 50 mm Tris-HCl buffer (pH 7.6). The resulting homogenate was frozen overnight at -20°C before centrifugation at 100,000g to obtain a pellet containing membranes.Solubilization of Lycopene Cyclase Activity. The above membrane fraction was dispersed in 20 ml of buffer solution containing 1 mM MgC92, 10 mm DTT, and 50 mm and quickly added to 200 ml of freshly distilled acetone kept at -20C. The aggregates were dispersed with a Teflon pestle. The solution was filtered within 1 min and the residue was extracted again using the same procedure. The residual acetone powder was washed twice with 50 ml of freshly distilled ether at -20°C. The dried powder was extracted with a buffer solution containing 5 mM DTT and 100 mm K2HPO4-KH2PO4 buffer (pH 7). After centrifugation at 100,000g for 4 h, the supernatant was used for enzymic determinations.

The Carotenoid Cleavage Dioxygenase 1 Enzyme Has Broad Substrate Specificity, Cleaving Multiple Carotenoids at Two Different Bond Positions

“…The loss of activity was not due to the buffer used to elute the protein as assays with ZmCCD1 lysate containing the elution buffer retained activity. Other membrane-bound and membrane-associated proteins show a similar loss of activity upon purification (45). Alternatively the GST tag could inhibit activity, but extremely low volatile levels from E. coli expressing native or His-tagged CCD enzymes argues against this possibility (data not shown).…”

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

“…Many attempts have been made to isolate enzymes that catalyze this reaction, and the work of Bramley and Taylor (1985), Dogbo (1986), andBeyer (1987) have demonstrated that detergent solubilization of these membrane-bound enzymes can be achieved. However, the activity is not particularly stable, and a purification to homogeneity has remained elusive.…”

“…Studies done with crudely purified extracts have been inconclusive in determining what, if any, are the cofactor requirements. Cyclization of lycopene in detergent extracts or acetone powders of Capsicurn annuum chromoplasts (Camara and Dogbo, 1986) was found not to require NADP+ or FAD, whereas tomato cellfree extracts absolutely required FAD and were stimulated by NADP+ (Kushwaha et al, 1969). A recent study examining the lycopene cyclase gene of E. herbicola (crtY) overexpressed in E. coli reported that only the all-trans isomer of lycopene was a substrate in crude cell-free lysates (Hundle et al, 1993).…”

Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation.

“…The enzymes themselves are also tightly associated with plastid membranes (Bramley, 1985;Camara and Dogbo, 1986;Beyer, 1987 are similar, and both contain several hydrophobic regions that could correspond to membrane-spanning helical domains. However, none of these regions reaches a magnitude of hydrophobicity (a value >1.6 for a window of 19 amino acid residues) that would indicate a high probability of forming a transmembrane helix, according to Kyte and Doolittle (1982).…”