Functional Elucidation Of Site 377 In Cryptochrome/Photolyase Family

The cryptochrome/photolyase family(CPF)is a protein family that is responsible for repairing UV-induced DNA lesions or regulating varieties of biochemical reactions in the living organisms.As the ubiquitous photoreceptors,CPF proteins play important roles in the living organisms by absorbing light energy,with flavin adenine dinucleotide(FAD)as a cofactor.Due to the functional diversity,CPF proteins are divided into the photolyases and the cryptochromes.Photolyases are DNA repair enzymes that absorb the light energy from 315 nm to 500 nm to repair DNA damage caused by exposure to ultraviolet(UV)light and to restore the normal structure of DNA.Two kinds of DNA lesions are induced by UV light,cyclobutane pyrimidine dimer(CPD)and pyrimidine(6-4)pyrimidone photoproduct(6-4photoproduct)which repress DNA replication and transcription.According to the repaired substrates,the photolyases can be further divided into CPD photolyases and 6-4 photolyases.Furthermore,CPD photolyases can further fall into two types,CPDI/III type and CPDII type,based on the homology of sequences.Cryptochromes are the proteins that share highly homologous sequences and structures with photolyases and usually lack DNA repair activity.They were first characterized for Arabidopsis thaliana and are also found to be ubiquitous in the plants and animals.Cryptochromes mediate a variety of light responses and are involved in the growth,development,circadian rhythm and navigation of many living organisms.There are two kinds of cryptochromes.Plant cryptochromes are photoreceptors that are involved in the blue-light-dependent development and growth of plants.Animal cryptochromes can be further divided into two types.Type I animal cryptochromes are light-dependent receptors for entrainment of the circadian rhythm,and Type II animal cryptochromes are light-independent regulatory components of the circadian clock.As the electron transfer mediator,FAD can be reduced or oxidated by one-and two-electron transfer reactions.FAD contains five forms,including fully oxidation,neutral blue radical,anionic red radical,neutral fully reduction,anionic fully reduction.Based on the analyses of sequence alignment and structure,it is found that a key residue displays an interesting distribution pattern in the CPF proteins.This key residue is A377 in Escherichia coli CPD photolyase,which is hereafter referred to as "site 377".CPD photolyases often have amino acids with small or polar side chains at this site,such as Ala,Ser and Asn.By contrast,most 6-4 photolyases and all animal cryptochromes have bulky hydrophobic residues at this site,such as Ile,Leu and Val.The residue is located in FAD binding pocket,suggesting that this site may have an influence on redox property or stability which finally affect the protein function.In this study,in order to investigate the functions of 377 site,E.coli CPD photolyase mutants(A377S,A377 N and A377I),Phaeodactylum tricornutum 6-4 photolyase mutants(I423A,I423 S and I423N)and Drosophila melanogaster cryptochrome mutants(V415S and V415N)were constructed by site-directed mutagenesis.SDS-PAGE showed that the molecular weight of purified mutant proteins from E.coli CPD photolyase,P.tricornutum 6-4 photolyase and D.melanogaster cryptochrome were about 50 k Da,61 k Da,60 k Da,respectively.Gel filtration chromatography showed the same results as SDS-PAGE.It is no doubt that these three CPF proteins are the monomeric state in solution.Comparing to the wild-type CPF proteins,UV-vis spectra revealed that the typical absorption peaks of FAD for the mutants with Ala,Ser and Asn at377 position were blue-shifted,whereas those peaks for the mutants with Ile,Leu and Val at 377 position were red-shifted.The shift in the spectral lines indicates that the binding pocket of FAD has been changed in these mutants,resulting the delocalization changes of the π-electron system of FAD isoalloxazine moiety.Photoreduction results showed that the mutants with Ala,Ser and Asn at 377 position yielded higher reduction reaction rate and the quantum production than those of the wild-type photolyases,whereas the reduction reaction rate and the quantum production of the mutants with Ile at377 position were decreased dramatically.Furthermore,the D.melanogaster cryptochrome mutants with Ser and Asn at 377 position(V415S and V415N)would further result in the formation of the fully reduced FAD(HQ).Oxidation kinetics desmonstrated that the mutants with Ala,Ser and Asn at site 377 decreased the oxidization rate of FAD with respect to the wild-type photolyases,whereas the mutants with Ile at this site increased the oxidated rate of FAD.Activity assays showed that the changes of Ala,Ser and Asn at377 position did not enhance the catalysis activity of CPF proteins,but resulting in the long-lasting activity due to the improved ability against oxidation.This phenomenon was evident for CPD photolyasesIn this study,it has been proved that the residues at 377 site are related with the regulation of HQ stability for the photolyases.HQ stability can be increased by CPD photolyase-prone residues such as Ala,Ser and Asn,but decreased by bulky hydrophobic residues such as Ile.Animal cryptochromes contain bulky hydrophobic residues at 377 site,which may be of some benefit for stablizing the ASQ or NSQ state to maintain the signal transduction.Indeed,the wild-type D.melanogaster cryptochrome can only be photoreduced to the ASQ state,but its mutants(V415S and V415N)(Val415is equivalent to 377 site)can be further photoreduced to the HQ state.Therefore,the results reveal that the occurrence of the bulky hydrophobic residues at 377 site could be an important evolutionary event fro CPF proteins,which is the supportive evidence for the evolution of animal cryptochromes from the photolyases.