Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD. (15th May 2017)

• Record Type: Journal Article
• Authors: Xu, Lei; Wen, Bin; Wang, Yuan; Tian, Changqing; Wu, Mingcai; Zhu, Guoping
• Is Part Of: Chembiochem. Volume 18:Number 12(2017)
• Page Start: 1129
• Page End: 1137
• Publication Date: 2017-05-15
• Subjects: cryptochromes -- flavin -- photochemistry -- photolyases; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201700145
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV‐induced cyclobutane pyrimidine dimers (CPDs) or 6–4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as "site 377", that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light‐dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases. Abstract : Single switches : Replacing Ala377 in E. coli photolyase with hydrophobic residues resulted in lower affinity for reduced FAD, whereas homologous Drosophila cryptochrome (dCRY) mutants with Ser or Asn substitutions at Val415 (corresponding to site 377) gave higher yields of reduced FAD during photoreduction. Site 377 substitutions might therefore confer different preferences for reduced FAD and enable differentiation.