April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Roles of Critical Aromatic Residues in RPE65 Retinol Isomerase Activity
Author Affiliations & Notes
  • T. M. Redmond
    LRCMB, National Eye Inst/NIH, Bethesda, Maryland
  • P. Chander
    LRCMB, National Eye Inst/NIH, Bethesda, Maryland
  • S. Gentleman
    LRCMB, National Eye Inst/NIH, Bethesda, Maryland
  • E. Poliakov
    LRCMB, National Eye Inst/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  T.M. Redmond, None; P. Chander, None; S. Gentleman, None; E. Poliakov, None.
  • Footnotes
    Support  NEI Intramural Research Program
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1213. doi:
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      T. M. Redmond, P. Chander, S. Gentleman, E. Poliakov; Roles of Critical Aromatic Residues in RPE65 Retinol Isomerase Activity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1213.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : RPE65, one of a family of carotenoid oxygenases, is established as the visual cycle retinol isomerase. The catalytic site of these enzymes, based on the crystal structure of Synechocystis apocarotenal oxygenase (ACO) contains 4 histidine residues coordinating ferrous iron and 3 fixing glutamates. Inter-strand and inter-blade loops cover the catalytic site and these are predicted to govern substrate specificity. Hydrophobic or aromatic residues in the substrate-binding tunnel are predicted to interact with the polyene substrate. Understanding these interactions may help decipher the mechanism of retinol isomerization by RPE65.

Methods: : RPE65 sequence was modeled on the ACO structure template to identify conserved aromatic residues that may interact with the substrate. Site-directed mutagenesis was used to make panels of mutants for several conserved aromatics. These were assayed for their effects on RPE65 isomerase activity. Expression levels of these mutant enzymes were quantitated by fluorescent immunoblotting.

Results: : Several aromatic residues line the predicted substrate cleft and may interact with substrate, including F61, Y239 and W331. The beta-ionone ring of the substrate was placed adjacent to the W331 sidechain. Mutation of Y239 to other aromatic or hydrophobic residues (W, F and L) or to hydroxyl/thiol residues (S, T and C) abolishes activity, as does Y239D, a human pathogenic mutation. Thus Y239 appears to be an essential residue. For F61, the mutants F61W and F61L are inactive, while F61Y has only 7.5% of wildtype activity. Mutants of W331 had the most interesting changes: W331Y (paralogous residue in beta-carotene monooxygenase (BCMO) 1) had 25% and W331F had 6% of wildtype activity, respectively; activities of W331L and W331Q (paralogous residue in BCMO2) were abolished.

Conclusions: : All three residues studied are required for RPE65 activity. F61 and, especially, Y239 are rather intolerant of change. However for W331, retention of aromaticity at this position is crucial to the catalytic activity of RPE65. If W331 interacts with the retinoid substrate, it may do so by cation-pi binding. In this regard, it is interesting that the graded pattern of relative activity of the mutants is consistent with the established pi-binding energy series of the aromatic residues, W>Y>F.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium 

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