Femtochemistry in enzyme catalysis: DNA photolyase

Kao, Ya-Ting; Saxena, Chaitanya; Wang, Limin; Sancar, Aziz; Zhong, Dongping

doi:10.1007/s12013-007-0034-5

Cited by 36 publications

(39 citation statements)

References 84 publications

(122 reference statements)

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“…A, the Trp triad. The arrows show the two pathways of photoreduction (16,17); that is, electron hopping through the Trp triad and electron tunneling through ␣-helix 15 and Phe-366. The Trp triad residues in Type 1 CRYs that correspond to the E. coli Trp triad and which were mutated in this study are indicated in parentheses.…”

Section: Purification Of Insect Typementioning

confidence: 99%

Animal Type 1 Cryptochromes

Öztürk

Song

Selby

et al. 2008

Journal of Biological Chemistry

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100

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It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD . , in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD ox when purified, and the flavin is readily reduced to FAD . by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD . in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD . represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.Cryptochromes are photolyase-related flavoproteins that play important roles in regulating the circadian clock in animals and growth and development in plants (1-3). The mechanism of photosignaling by animal cryptochromes is not known. Previously, it was thought that CRYs 3 in Drosophila and other insects function as circadian photoreceptors and in mouse and other vertebrates function as core components of the molecular clock (4). Recently, this view was revised when it was realized that some insects such as the honeybee possess only a mammalian CRY-like cryptochrome and others such as the monarch butterfly possess both Drosophila CRY-like and mammalian CRY-like cryptochromes (5, 6). It was proposed that Drosophila-like CRYs should be referred to as Type 1 CRYs and the mammalian-like CRYs should be referred to as Type 2 CRYs (6). Furthermore, it was found that all Type 1 CRYs tested were subject to light-induced proteolysis in Schneider 2 (S2) cells and, hence, were considered to function as circadian photoreceptors in a manner analogous to DmCRY (6). Similarly, it was shown that insect Type 2 CRYs, like the mammalian CRYs, functioned as core clock proteins with no demonstrable photoreceptor activity (6).We are interested in the photoreceptor function of CRY and specifically in the cryptochrome photocycle. Type 1 CRYs are well suited for this purpose because their photoinitiated proteolysis constitutes a convenient functional assay (7-9). Two recent studies reported that Type 1 CRYs from Drosophila melanogaster and the monarch butterfly (Danaus plexippus), purified as recombinant proteins, contained near-stoichiometric amounts of flavin in the two-electron oxidized, FAD ox , form. Exposure of these CRYs to light reduced the flavin to the flavin anion ...

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Section: Purification Of Insect Typementioning

confidence: 99%

Animal Type 1 Cryptochromes

Öztürk

Song

Selby

et al. 2008

Journal of Biological Chemistry

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100

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“…CPD can be completely restored by a photoenzyme, photolyase, through absorption of visible blue light (4). In our early work (5-7), we have observed a cyclic electron-transfer (ET) reaction in thymine dimer (ThiT) repair by photolyase and determined the time scale of 700 ps for the complete repair photocycle (7). However, the central questions of whether the splitting of the cyclobutane ring is synchronously or asynchronously concerted or stepwise and whether the cyclic ET involves specific tunneling pathways were not resolved.…”

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confidence: 99%

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Liu

Tan

Guo

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme's high repair efficiency.DNA repair photocycle | ultrafast enzyme dynamics | thymine dimer splitting | electron tunneling pathway | active-site mutation U ltraviolet (UV) component of sunlight irradiation causes DNA damage by inducing the formation of cyclobutane pyrimidine dimer (CPD), which is mutagenic and a leading cause of skin cancer (1-3). CPD can be completely restored by a photoenzyme, photolyase, through absorption of visible blue light (4). In our early work (5-7), we have observed a cyclic electron-transfer (ET) reaction in thymine dimer (ThiT) repair by photolyase and determined the time scale of 700 ps for the complete repair photocycle (7). However, the central questions of whether the splitting of the cyclobutane ring is synchronously or asynchronously concerted or stepwise and whether the cyclic ET involves specific tunneling pathways were not resolved. Furthermore, the molecular mechanism underlying the high repair efficiency has not been elucidated. Here, using femtosecond spectroscopy and site-directed mutagenesis, we are able to measure the dynamics of all initial reactants, reaction intermediates, and final products with different substrates and with wild-type and active-site mutant enzymes, and thus reveal the complete spatio-temporal molecular picture of thymine dimer repair by photolyase.Photolyase contains a fully reduced flavin adenine dinucleotide (FADH − ) as the catalytic cofactor and electron donor (4). Based on previous studies (4-10), a sequential repair mechanism of thymine dimer splitting is shown in Fig. 1. Previously, we found that the forward ET from FADH − Ã to ThiT occurs in 250 ps (1∕k FET ) and the total decay of intermediate FADH• in 700 ps (1∕k total ) (5, 7). These dynamics usually follow a stretched-exponential decay behavior, reflecting heterogeneous ET dynamics controlled by the active-site solvation (5, 6, 11). However, in that study, no thymine-related species could be detected in the vi...

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“…Thus, upon excitation the local polar environments of these function sites would proceed to a series of relaxations. Understanding of these local motions is crucial to their functional dynamics of resonance energy transfer from the photoantenna to the cofactor and electron transfer between the cofactor and the substrate in catalytic repair photocycle.Our earlier studies on Escherichia coli CPD photolyase (EcPhr) showed that the active-site solvation is on the similar time scale of the catalytic reaction of electron transfer in hundreds of picoseconds, resulting in a nonequilibrium dynamic process and a direct modulation of charge separation (10,(27)(28)(29). The characterization of resonance energy transfer from the photoantenna (30, 31) revealed minor solvation at the binding site in less than 20 ps.…”

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confidence: 99%

“…Our earlier studies on Escherichia coli CPD photolyase (EcPhr) showed that the active-site solvation is on the similar time scale of the catalytic reaction of electron transfer in hundreds of picoseconds, resulting in a nonequilibrium dynamic process and a direct modulation of charge separation (10,(27)(28)(29). The characterization of resonance energy transfer from the photoantenna (30, 31) revealed minor solvation at the binding site in less than 20 ps.…”

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confidence: 99%

Ultrafast solvation dynamics at binding and active sites of photolyases

Chang

Guo

Kao

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Dynamic solvation at binding and active sites is critical to protein recognition and enzyme catalysis. We report here the complete characterization of ultrafast solvation dynamics at the recognition site of photoantenna molecule and at the active site of cofactor/ substrate in enzyme photolyase by examining femtosecondresolved fluorescence dynamics and the entire emission spectra. With direct use of intrinsic antenna and cofactor chromophores, we observed the local environment relaxation on the time scales from a few picoseconds to nearly a nanosecond. Unlike conventional solvation where the Stokes shift is apparent, we observed obvious spectral shape changes with the minor, small, and large spectral shifts in three function sites. These emission profile changes directly reflect the modulation of chromophore's excited states by locally constrained protein and trapped-water collective motions. Such heterogeneous dynamics continuously tune local configurations to optimize photolyase's function through resonance energy transfer from the antenna to the cofactor for energy efficiency and then electron transfer between the cofactor and the substrate for repair of damaged DNA. Such unusual solvation and synergetic dynamics should be general in function sites of proteins.function-site solvation | ultrafast dynamics | spectral tuning | protein rigidity and flexibility | femtosecond-resolved emission spectra D ynamic solvation in binding and active sites plays a critical role in protein recognition and enzyme reaction, and such local motions optimize spatial configurations and minimize energetic pathways (1-11). These dynamics involve local constrained protein and trapped-water motions within angstrom distance and occur on ultrafast time scales (6,7,10,11). Typically, extrinsic dye molecules or synthetic amino acids were used as local optical probes to label function sites, and the local relaxations were observed, ranging from femtoseconds to nanoseconds (5,(12)(13)(14). Such labeling of bulky dye molecules usually induces significant local perturbations, and direct characterization with intrinsic chromophores in proteins eliminates those interferences and reveals intact environment responses (4, 7, 15-21), as recently examined in green fluorescence proteins (21). We have recently studied a series of flavoproteins using intrinsic flavin molecule as the optical probe (10, 22, 23) and especially found the important functional role of local solvation in photolyase (10).Photolyase, a flavoprotein and a photoenzyme, repairs damaged DNA caused by UV irradiation. Two types of structurally similar photolyases are specific for two major UV-induced DNA lesions of cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (24). The CPD photolyase contains two noncovalently bound chromophores, a pterin molecule in the form of methenyltetrahydrofolate (MTHF) in the binding site as the photoantenna for energy efficiency and a fully reduced flavin adenine dinucleotide (FADH − ) at the active site as the catalytic cofactor for repair of d...

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Femtochemistry in enzyme catalysis: DNA photolyase

Cited by 36 publications

References 84 publications

Animal Type 1 Cryptochromes

Animal Type 1 Cryptochromes

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Ultrafast solvation dynamics at binding and active sites of photolyases

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