Impacts of Cys392, Asp393, and ATP on the FAD Binding, Photoreduction, and the Stability of the Radical State of Chlamydomonas reinhardtii Cryptochrome


Abstract

Plant cryptochromes (CRYs) are blue-light receptors that regulate the light-dependent growth, development and circadian rhythm. A flavin adenine dinucleotide (FAD) cofactor is bound to the photolyase homology region (PHR) of plant CRYs that can be photoreduced to neutral radical state under blue light. This photoreaction may trigger subsequent signal transduction. Plant CRYs can also bind an ATP adjacent to FAD in a pocket of PHR. Chlamydomonas reinhardtii contains a single plant CRY, named Chlamydomonas photolyase homologue 1 (CPH1). In CPH1, Cys392 and Asp393 is located near the FAD cofactor. Here we showed that replacing Cys392 with Ser had little effect on the properties of CPH1. But the C392N mutant showed a faster photoreduction rate, and a significantly lower oxidation rate of the neutral radical state compared with those of wild type CPH1. Substituting an Asn for Asp393 in CPH1 improved the binding affinity for FAD as well as the stability of the neutral radical; but photoreduction of the mutant was severely inhibited. In the presence of ATP, CPH1 and its mutants exhibited significantly higher binding affinity for FAD and slower oxidation of the neutral radical. These results reveal that the residues at site 392 and the presence of ATP can tune the stability of the neutral radical; the Asp at site 393 is crucial for photoreduction; and the photoreduction rate was not merely determined by the stability of the neutral radical in CPH1.

Submission Details

ID: WCoru98s3

Submitter: Lei Xu

Submission Date: Dec. 13, 2018, 5:31 p.m.

Version: 1

Publication Details
Lei Xu, Bin Wen, Wengui Shao, Pengcheng Yao, Wei Zheng, Zhiqiang Zhou, Yao Zhang, and Guoping Zhu,Chembiochem (2018) Impacts of Cys392, Asp393, and ATP on the FAD Binding, Photoreduction, and the Stability of the Radical State of Chlamydomonas reinhardtii Cryptochrome
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