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.
Submitter: Lei Xu
Submission Date: Dec. 13, 2018, 5:31 p.m.
|Number of data points||48|
|Assays/Quantities/Protocols||Experimental Assay: Oxidation without ATP ; Experimental Assay: Photoreduction without ATP ; Experimental Assay: The apparent Kd with ATP ; Experimental Assay: UV-vis absorption spectroscopy ; Experimental Assay: The apparent Kd without ATP ; Experimental Assay: Photoreduction with ATP ; Experimental Assay: Oxidation with ATP ; Experimental Assay: photorepair activity ; Derived Quantity: SD of The apparent Kd without ATP ; Derived Quantity: SD of The apparent Kd with ATP ; Derived Quantity: SD of Photoreduction with ATP ; Derived Quantity: SD of Oxidation with ATP ; Derived Quantity: SD of Photoreduction without ATP ; Derived Quantity: SD of Oxidation without ATP|
|Libraries||Variants for CPH1-FAD complex ; Variants for CPH1|