Purchase this article with an account.
Preethi Chander, Susan Gentleman, Eugenia Poliakov, T. Michael Redmond; Phenylalanines Have Distinct Effects On Rpe65 Isomerase Activity. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3330.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
RPE65 retinol isomerase is a member of the carotenoid oxygenase family, recent crystal structures of which reveal a common 7-bladed beta-propeller fold with helices and loops on one side of the propeller. Placement of essential iron-coordinating histidines and fixing glutamates are virtually identical in all crystal structures. Other residues lining the hydrophobic substrate cleft, predominantly aromatic, are also conserved. Identifying their role in isomerase activity will help discern the underlying mechanism of RPE65.
Based on the structures of RPE65, ACO and VP14, a model of the substrate-bound form of RPE65 was built. A rational mutagenesis approach was taken to make mutants of invariant Phe residues (61, 312 and 526) which were assayed for isomerase activity and expression levels.
The substrate cleft was observed to be mainly hydrophobic; residues lining the substrate cleft were predominantly aromatic. F526, 312 and 61 were located in the vicinity of the substrate ester bond. Replacement of F526 with other aromatic or hydrophobic residues severely reduced (Y and W) or abolished (L) isomerase activity. Interestingly, F526A showed ~40% increased levels of 13-cis retinol production relative to wildtype (WT), while 11-cis production was reduced by a similar amount. Protein levels for the null mutants were ~50% WT; while that of F526A was at par with WT. In ACO and BCMO1 this position is occupied by leucine. For residue F61, L and W mutants were inactive and the Y mutant showed 14% activity relative to WT, while for F312, all mutants (L, Y and W) were inactive.
Aromatic residues play an important role in isomerase activity of RPE65. The Phe residues investigated here play a specific role due to their chemistry and vicinity to the substrate ester bond. Effects of F526 mutation suggest a pi-pi interaction with the aromatic sidechain of F312. We propose that these disturb the delocalization of charges on the polyene substrate/intermediate chain, thus impacting chemistry around the ester bond. We previously showed that F103 also displays altered 11-cis/13-cis ratio. However, in contrast to F526, all F103 mutants showed altered product ratio. F103 likely plays a role in specificity by determining substrate/intermediate orientation.
This PDF is available to Subscribers Only