Abstract
Purpose :
When light enters a animal’s eye, there is a retinal light response that initiates from photoisomerization of an opsin-coupled 11-cis-retinaldehyde chromophore. Carotenoids are important precursors to produce the visual chromophore, 11-cis-retinal, through the action of carotenoid cleavage dioxygenases (CCDs). CCDs are non-heme iron enzymes that catalyze the oxidative cleavage of carbon-carbon double bonds in carotenoid backbones, generating aldehyde or ketone groups at the cleavage ends. Vertebrates express two different CCDs, BCO1, which catalyzes its reaction as an oxygenase and RPE65, which catalyzes its reaction as an isomerase. Interestingly, in invertebrates there is a single enzyme that contributes to the formation of the 11-cis-retinaldehyde chromophore known as NinaB. NinaB not only cleaves carotenoids symmetrically but also isomerizes one-half of the substrate to form an 11-cis retinaldehyde product. NinaB is termed an “isomerooxygenase” after its dual activity. We aim to report our new understanding of the mechanism of action for the dual functioning isomerooxygenase, NinaB.
Methods :
Through a large ortholog screen, we have identified a NinaB protein exhibiting high-level soluble expression in E. coli. As the next step, we developed a robust purification method, resulting in pure, catalytically active protein against substrates beta-carotene and zeaxanthin. Using our x-ray crystallographic structure, and structure-guided mutagenizes of NinaB, allowed us to screen residues critical for the isomerization function of NinaB. To confirm the importance of membrane-binding residues, we expressed mutants in carotenoid-producing E. coli and visually inspected cleavage efficiency of carotenoids.
Results :
We report the structure of NinaB at a resolution of 1.9 Å revealing details of its active site architecture and mode of membrane binding. We have identified a residue cluster deep within the NinaB substrate-binding cleft important for its isomerization activity and membrane binding.
Conclusions :
Comparative enzymology allows us to bridge a gap of understanding vertebrate and invertebrate carotenoid metabolism. At a molecular level, we have demonstrated that isomerization activity is mediated by different active site regions in NinaB and RPE65. In addition, by accomplishing the x-ray crystallographic structure of NinaB, we have added to our prior understanding of RPE65.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.