Identification of catalytically important residues of the carotenoid 1,2-hydratases from Rubrivivax gelatinosus and Thiocapsa roseopersicina.

Carotenoid 1,2-hydratases (CrtC) catalyze the selective addition of water to an isolated carbon-carbon double bond. Although their involvement in the carotenoid biosynthetic pathway is well understood, little is known about the mechanism by which these hydratases transform carotenoids such as lycopene into the corresponding hydroxyl compounds. Key residues were identified at positions His239, Trp241, Tyr266, and Asp268 in CrtC from Rubrivivax gelatinosus (and corresponding positions in Thiocapsa roseopersicina).

Enzymatic hydration activity assessed by selective spectrophotometric detection of alcohols: a novel screening assay using oleate hydratase as a model enzyme.

Hydroxy fatty acids (HFAs) are high-added-value compounds, which are incorporated in polymers, lubricants, emulsifiers and stabilizers and have potential medicinal use. In nature, HFAs are regio-specifically synthesized by several enzymes, including P450 monooxygenases, lipoxygenases, hydratases, 12-hydroxylases, and diol synthases. The growing demand for HFAs warrants the development of simple and efficient analytical methods that enable high-throughput detection of the hydroxylated product in the presence of its unsaturated precursor.

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

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.