Role of Tyrosines in Mouse β‐Carotene 15,15′‐Monooxygenase Activity

Abstract

β-carotene monooxygenase (BCMO1) is a key enzyme in vitamin A biosynthesis in mammals. It symmetrically cleaves β-carotene to two molecules of all trans-retinal. The process of monooxygenation involves reductive oxygen activation at metal complexes and then oxygen transfer to substrate. Tyrosines play a key role in coupled proton-electron transfer in a large number of metalloenzymes. Therefore, we decided to investigate the role of tyrosines conserved among mammalian family members by oligonucleotide-directed mutagenesis. We found that while activities of Y235F, Y236F, Y418F, Y420F mutants of mouse BCMO1 were reduced, they all retained a substantial activity (more than 60% of wt activity). While change of Y235 and Y420 to leucines led to significant impairment of activity (less than 35% of wt), change of Y411, Y413 and Y418 to non-aromatic amino acids did not have a strong effect on BCMO1 activity. We also replaced the non-conserved Y326, predicted from our model to be located in the binding tunnel of mouse BCMO1. Changing to an aromatic (Y326W) had little influence on enzyme activity, while change to a non-aromatic residue (Y326L) substantially reduced activity. Therefore, we conclude that while the studied tyrosines are not essential for the catalytic mechanism, it seems that the presence of aromatic rings at Y235, Y326, Y420 is important for BCMO1 activity and they could be involved in electron transfer in BCMO1.