Cloning and Functional Analysis of the β-Carotene Hydroxylase of Arabidopsis thaliana

An Arabidopsis thaliana cDNA encoding the enzyme β-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both β rings of the symmetrical β-carotene (β,β-carotene) to form zeaxanthin (β,β-carotene-3,3′-diol) and converts the monocyclic β-zeacarotene (7′,8′-dihydro-β,ψ-carotene) to hydroxy-β-zeacarotene (7′,8′-dihydro-β,ψ-carotene-3-ol). The ϵ rings of δ-carotene (ϵ,ψ-carotene) and α-zeacarotene (7′,8′-dihydro-ϵ,ψ-carotene) are poor substrates for the enzyme. The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bacterial β-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E. coli. Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate β-cryptoxanthin (β,β-carotene-3-ol), predominantly, when β-carotene was provided as the substrate. It is suggested that amino acid residues 70-129 of the A. thaliana enzyme may play a role in formation of a functional homodimer. An Arabidopsis thaliana cDNA encoding the enzyme β-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both β rings of the symmetrical β-carotene (β,β-carotene) to form zeaxanthin (β,β-carotene-3,3′-diol) and converts the monocyclic β-zeacarotene (7′,8′-dihydro-β,ψ-carotene) to hydroxy-β-zeacarotene (7′,8′-dihydro-β,ψ-carotene-3-ol). The ϵ rings of δ-carotene (ϵ,ψ-carotene) and α-zeacarotene (7′,8′-dihydro-ϵ,ψ-carotene) are poor substrates for the enzyme. The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bacterial β-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E. coli. Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate β-cryptoxanthin (β,β-carotene-3-ol), predominantly, when β-carotene was provided as the substrate. It is suggested that amino acid residues 70-129 of the A. thaliana enzyme may play a role in formation of a functional homodimer.