Enzymatic isomerization of ζ-carotene mediated by the heme-containing isomerase Z-ISO

Carotenoids are a large and diverse class of isoprenoid compounds synthesized by plants, algae, some bacteria, arthropods, and fungi. These pigments contribute to plant growth and survival by protecting plants from photooxidative stress and serving as precursors of plant hormones and other signaling compounds. In humans, carotenoids are essential components of the diet and contribute anti-oxidant and provitamin A activities. Carotenoids are synthesized in the membranes of plant plastids where phytoene is converted into all trans lycopene by a biosynthetic pathway that was only recently completed by the discovery of the new enzyme, 15-cis-ζ-carotene isomerase (Z-ISO), which controls carotenoid pathway flux to products necessary for plant development and function. Z-ISO catalysis of the cis to trans isomerization of the 15-cis double bond in 15-cis-ζ-carotene is mediated by a unique mechanism dependent on the redox-state of a heme b cofactor. This chapter describe methods for the functional analysis of Z-ISO, including complementation of Z-ISO in engineered E. coli, separation of Z-ISO enzyme substrate and products, ζ-carotene isomers, by high pressure liquid chromatography (HPLC), expression and purification of Z-ISO and in vitro enzymatic reactions.