Abstract
CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C40 hydrocarbon substrate.
Highlights
Carotenoids are indispensible in photosynthetic energy metabolism both, in prokaryotes and eukaryotes, where they serve in light harvesting and photoprotection
Cyanobacteria and plants employ a complex, multi-component pathway relying on two desaturases, namely phytoene desaturase (PDS) and fcarotene desaturase (ZDS), that form specific poly-cis configured carotene intermediates [5,6] necessitating the participation of two cistrans isomerases
CRTI Purification and Enzymatic Activity Overexpression in E. coli produced a substantial proportion of CRTI-His6 soluble protein allowing purification to near homogeneity by IMAC and subsequent gel permeation chromatography (GPC; Figure 2), where the protein eluted at the position expected for the monomeric form (56.04 kDa)
Summary
Carotenoids are indispensible in photosynthetic energy metabolism both, in prokaryotes and eukaryotes, where they serve in light harvesting and photoprotection. B-carotene and oxygenated xanthophylls serve in plants as precursors in the formation of phytohormones, such as abscisic acid and the strigolactones [1,2]. Carotenoids are colored due to their polyene chromophore These (mostly) eleven conjugated double bonds are formed from saturated precursors by carotene desaturases. Cyanobacteria and plants employ a complex, multi-component pathway relying on two desaturases, namely phytoene desaturase (PDS) and fcarotene desaturase (ZDS), that form specific poly-cis configured carotene intermediates [5,6] necessitating the participation of two cistrans isomerases.
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