CLONING AND FUNCTIONAL-ANALYSIS OF THE BETA-CAROTENE HYDROXYLASE OF ARABIDOPSIS-THALIANA

An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxy lase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both beta rings of t he symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin ( beta,beta-carotene-3,3'-diol) and converts the monocyclic beta-zeacaro tene (7',8'-dihydro-beta,psi-carotene) to hydroxy-beta-zeacarotene (7' ,8'-dihydro-beta,psi-carotene-3-ol). The epsilon rings of delta-carote ne (epsilon,psi-carotene) and alpha-zeacarotene (7',8'-dihydro-epsilon ,psi-carotene) are poor substrates for the enzyme, The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bact erial beta-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Tru ncation of the cDNA to produce a polypeptide lacking the first 69 amin o acids does not impair enzyme activity in E. coli. Truncation to yiel d a polypeptide of a length comparable with the bacterial enzymes (lac king 129 N-terminal amino acids) resulted in the accumulation of the m onohydroxy intermediate beta-cryptoxanthin (beta,beta-carotene-3-ol), predominantly, when beta-carotene was provided as the substrate. It is suggested that amino acid residues 70-129 of the A. thaliana enzyme m ay play a role in formation of a functional homodimer.