The visual cycle, crucial for human vision, involves the recycling of the visual chromophore within the retina. This cycle enables us to process light and generate sight, granting humans and animals the ability to perceive their surroundings. Unfortunately, certain genetic conditions lead to visual diseases and loss of vision. Understanding the molecular mechanisms of key enzymes in this cycle, particularly those involved in visual chromophore production, has allowed scientists and clinicians to develop therapies aimed at preserving vision and managing these diseases. Conversely, a delayed understanding of key enzymes has inhibited the production of therapies. This thesis explores both vertebrate and invertebrate systems, particularly focusing on visual chromophore-producing enzymes to strengthen our understanding of the visual process across species.In animals, the retinal light response begins with the photoisomerization of the opsin-coupled 11-cis-retinaldehyde chromophore. This visual chromophore is enzymatically produced by carotenoid cleavage dioxygenases. In vertebrates, two such enzymes—β-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65)—convert carotenoid substrates into 11-cis-retinaldehyde. In contrast, invertebrates, such as insects, rely on a single enzyme, Neither Inactivation Nor Afterpotential B (NinaB), to perform this conversion. Both RPE65 and NinaB couple trans–cis isomerization with hydrolysis and oxygenation, respectively, but the detailed mechanism of their isomerase activities is still not fully understood. In this thesis, we present the structure of NinaB, shedding light on its active site and membrane-binding properties. Through structure-guided mutagenesis, we identify key residues in the substrate-binding cleft that regulate NinaB's isomerization activity. Our findings demonstrate that isomerization is mediated by distinct active site regions in NinaB and RPE65, providing a deeper understanding of the evolutionary convergence in visual system functions.
Another important enzyme in the visual cycle and retinoid processing in humans is lecithin:retinol acyl transferase (LRAT), which esterifies retinoids for storage in various tissues. While much is known about the enzymatic function of LRAT, there is still a lack of structural insight into this enzyme. Mutations in genes such as LRAT contribute to retinal diseases like retinitis pigmentosa (RP). Gaining a comprehensive structural understanding of LRAT could pave the way for therapeutic developments to treat such conditions. By exploring various biochemistry methods, we aim to enhance the current handling of the membrane protein LRAT for future studies.
Enzymes are typically classified into one of seven basic reaction classes, each corresponding to a specific type of chemical reaction. However, some enzymes catalyze multiple reaction types within a single active site. An example of this are isomerohydrolases and isomerooxygenases, which catalyze isomerization-coupled reactions essential for the production of 11-cis-retinoids, as mentioned above. In these enzymes, isomerization is coupled with other reactions, such as hydrolysis and oxygenation. A small number of enzymes exhibit similar coupled isomerization activities, some of which have been studied in detail. In an Appendix chapter, we also review these unique enzymes, exploring the structural and mechanistic basis of their reaction coupling, and revealing key catalytic commonalities that deepen our understanding of the visual cycle.