Key Role of Conserved Histidines in Recombinant Mouse β-Carotene 15,15′-Monooxygenase-1 Activity

Abstract

Alignment of sequences of vertebrate 405 , and Glu 457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate nonconserved in the overall family with alanines by sitedirected mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu 405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V max . The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu 405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase. ␤-Carotene 15,15Ј-monooxygenase-1 (BCMO1)1 is the initial enzyme step in the biosynthesis of vitamin A in animals, symmetrically cleaving ␤-carotene to produce two molecules of alltrans-retinal (1). In the last several years, a number of BCMO1 proteins have been cloned and biochemically characterized (2-7). BCMO1 belongs to a family of oxygenases of diverse activities, including lignostilbene dioxygenase in bacteria (8 -10); an epoxycarotenoid-cleaving enzyme required for abscisic acid biosynthesis in plants (11); and mammalian RPE65, a retinal pigment epithelial protein required for the production of the visual chromophore 11-cis-retinal in the visual cycle (12). The most recent addition to the characterized carotenoid oxygenases is cyanobacterial apocarotenoid 15,15Ј-oxygenase PCC 6803 (ACO) (13). Despite their unique functionalities and the obvious importance of these proteins, structural studies on this family have been hindered by technical difficulties, and only recently has the crystal structure of eubacterial ACO been published (14).Originally, Drosophila BCMO1 was identified by homology to a previously characterized plant 9-cis-epoxycarotenoid dioxygenase (15) and by its ability to cleave ␤-carotene (4). Subsequently, mouse BCMO1 was identified by our laboratory based on its homology to mammalian RPE65 (3). Because the biochemical function and mechanism of BCMO1 have been studied in detail (3, 5, 7), it is functionally the best characterized mammalian member of the family and could serve as a model for studying putative vertebrate oxygenases of more elusive function such as RPE65 and BCMO2 (16).Although there is a weak overall identity (Ͻ10%) am...

Optimized Formation of Detergent Micelles of β-Carotene and Retinal Production Using Recombinant Human β,β-Carotene 15,15'-Monooxygenase

“…These micelles are solubilized under aqueous conditions and are absorbed by intestinal cells (1)(2)(3)(4)(5)(6). Moreover, detergent micelles of -carotene have been used in the conversion of -carotene to retinal under aqueous conditions (4,10,(14)(15)(16)(17)(18)(19)(20). Thus, we investigated the effect of factors such as solvent, detergent, and substrate on the formation of detergent micelles of -carotene for retinal production under aqueous conditions.…”

“…However, because these micelles were used to investigate enzyme characterization other than retinal production, only a small amount of -carotene was used (4,10,(14)(15)(16)(17)(18)(19)(20).…”

Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle

“…We show that cotransfection of RPE65 and LRAT supports high-level synthesis of 11-cis-retinol. The abolition of 11-cis-retinol synthesis by mutation of key residues of RPE65, homologous to catalytic iron-coordinating residues in bona fide enzyme paralogs (22,27), strongly argues for a catalytic role for RPE65 in isomerization. Inhibition of isomerization by a chelator of ferrous iron further implicates catalytic iron.…”

“…4A, mutation to alanine of the conserved histidine residues (H180, H241, H313, and H527), paralogous to the iron-binding histidines of BCMO1, severely reduced or abolished 11-cis-retinol synthesis in cells transfected with these mutants compared with wild-type-transfected cells. E417A was also equally inactive; its paralog in BCMO1 (E405) is also involved in iron binding (27). In addition, E417Q, associated with retinal dystrophy (9), lacked activity (Table 1).…”

“…Alternatively, because it belongs to a family of carotenoid oxygenases, RPE65 may play a direct catalytic role in the visual cycle. This family, including bacterial (22), plant (23), invertebrate (24), and vertebrate (25, 26) carotenoid oxygenases, shares four absolutely conserved histidines that coordinate the iron required for activity of these enzymes (22,27). Because RPE65 retains these histidines, it might also bind catalytic iron to play a role in isomerization.…”