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Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase

Significance The formation of apocarotenoids by carotenoid cleavage dioxygenases (CCDs) is a critical process for several biological signaling systems. However, the active site determinants directing CCDs to cleave a specific alkene bond within the polyene backbone of carotenoids have remained unclear. Through analysis of the previously uncharacterized group of archaeal CCDs, we identified an enzyme, closely related to animal CCDs, that could be isolated in complex with its apocarotenoid product. Its crystal structure revealed the precise molecular interactions governing the enzyme’s unique regioselectivity. These insights pave the way toward control of CCD activity in carotenoid/retinoid metabolism through rational design of small-molecule modulators or targeted mutagenesis. Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD–carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14′–C13′ alkene bond to produce β-apo-14′-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.