Regulation of light harvesting in plants: structural and functional studies of the Violaxanthin De-epoxidase

Plants are sessile organisms: when environmental conditions change, they cannot move but have to respond by adapting their metabolism. This is particularly challenging with respect to illumination: harvesting efficiency must be maximized when light is limiting while in high light conditions all energy in excess has to be dissipated to avoid the fast formation of radical species harmful for the cell. Carotenoids are molecules with a key role in the response to environmental conditions, being able to safely dissipate potentially reactive excited species. In addition to this general ability of all carotenoids, when illumination is in excess there is the activation of the synthesis of zeaxanthin, a carotenoid particularly efficient in photoprotection. When light is back to normal, zeaxanthin is back converted into violaxanthin. Depending on illumination conditions, thus, there is a conversion between these two species, forming the so called xanthophyll cycle, which are modulating the light harvesting efficiency of photosynthetic apparatus. Enzymes involved in the xanthophyll cycle are the violaxanthin de-epoxidase (VDE) and zeaxanthin Epoxidase (ZE), which are catalyzing respectively the synthesis and degradation of zeaxanthin. The aim of this work was to understand the molecular mechanisms of the regulation of the xanthophyll cycle, by studying the biochemical and structural properties of these two enzymes. The attention was particularly focused on VDE, while only preliminary work was done on ZE (appendix 1). In the case of VDE, instead, we were able to obtain the first structural information on the enzyme, resolving the 3D structure of the lipocalin like domain of the protein, representing half of the full protein. Thanks to these data we were able to show how the protein in undergoing a pH dependent conformational change. Structural data also provided suggestions on the violaxanthin binding site to the enzyme. This hypothesis was tested in the chapter 2 by site directed mutagenesis. Merging structural and mutational data we were able to propose a confident model of violaxanthin binding to the enzyme, also identifying residues involved in the catalytic reaction. Finally in chapter 3 we analyzed the role of the remaining part of the protein for the protein function: major result was the finding that the C terminal domain, rich in Glu residues, is important for the association of the enzyme to the thylakoids membrane. In appendix 2 is instead presented some preliminary work on lycopene β-cyclase, an enzyme of the carotenoid biosynthetic pathway. We expressed and purified the recombinant protein to produce a specific antibody recognizing the enzyme as part of a wider project aiming to study the regulation of carotenoid biosynthesis in plants in response to environmental conditions.