Loss of Function in Zeaxanthin Epoxidase of <i>Dunaliella tertiolecta</i> Caused by a Single Amino Acid Mutation within the Substrate-Binding Site
Minjae Kim | Jisu Kang | Yongsoo Kang | Beom Sik Kang | EonSeon Jin
The <i>zea1</i> mutant of marine microalga <i>Dunaliella tertiolecta</i> accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type <i>D. tertiolecta</i> and the mutant. We found that the <i>zea1</i> mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and <i>zea1</i> were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in <i>zea1</i> was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of <i>zea1</i>, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.
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