Mechanism of Lutein to meso -Zeaxanthin Isomerization by RPE65 Catalysis

The macular pigments lutein (L), zeaxanthin (Z), and meso -zeaxanthin (MZ) protect the human retina from light and oxidative stress. While L and Z are abundant in the human diet, MZ is nearly absent. We previously demonstrated MZ is derived from L in chicken embryos precisely timed with expression of RPE65. Herein, we show that RPE65 from mouse, an animal that does not concentrate MZ in the eye, catalyzes L to MZ isomerization similarly as found for RPE65 from chicken and human, when expressed in cultured cells. Co-expression with xanthophyll-binding proteins had no impact on MZ yield. L and MZ both fit deep into the tunnel accessing the non-heme iron center, with strain evident for the 3’R , 6’R ε ring of L. Interestingly, a negatively charged Glu148, found along substrate tunnel, highly conserved among carotenoid cleavage dioxygenases, and which is critical for eye health, could be replaced by a neutral, isosteric residue (Gln) without impacting MZ yield. We propose that L to MZ isomerization proceeds by a neutral, radical transition state that differs from the carbocation encountered during retinoid isomerization. These findings extend our mechanistic understanding for macular carotenoid metabolism and should be considered when developing therapeutic interventions that act via RPE65.