Active Site of <i>Escherichia coli</i> DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair

Xu, Lei; Mu, Wanmeng; Ye, Ding; Z, Luo; Han, Qingkai; Bi, Fangfang; Wang, Yuzhen; Song, Qin‐Hua

doi:10.1021/bi800391j

Cited by 30 publications

(40 citation statements)

References 38 publications

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“…the N5-proximal residue should have a large affect on the FAD redox properties; indeed, mutation of this Asn to Ser in E. coli PL also severely destabilized the sq (39). However, the results with the Ser mutant do not establish how the conserved Asn stabilizes the neutral sq in wild type CPD-PL.…”

Section: Resultsmentioning

confidence: 88%

“…In CPD-PL, the N5-proximal Asn is not likely to act as a proton shuttle given that in solution it is very difficult to protonate (pK a Ͻ0) or deprotonate (pK a ϳ17). Instead, it has been proposed to stabilize the neutral sq radical by providing its side chain carbonyl as an H-bond acceptor (39). As in flavodoxins, full oxidation of CPD-PL requires sq deprotonation and loss of this H-bond, unless the Asn flips orientation to present its side chain amino groups as an H-bond donor to the N5.…”

Section: Resultsmentioning

confidence: 99%

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Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

Damiani

Nostedt

O’Neill

2011

Journal of Biological Chemistry

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Flavoproteins can dramatically adjust the thermodynamics and kinetics of electron transfer at their flavin cofactor. A versatile regulatory tool is proton transfer. Here, we demonstrate the significance of proton-coupled electron transfer to redox tuning and semiquinone (sq) stability in photolyases (PLs) and cryptochromes (CRYs). These light-responsive proteins share homologous overall architectures and FAD-binding pockets, yet they have evolved divergent functions that include DNA repair, photomorphogenesis, regulation of circadian rhythm, and magnetoreception. We report the first measurement of both FAD redox potentials for cyclobutane pyrimidine dimer PL (CPD-PL, Anacystis nidulans). These values, E 1 (hq/sq) ‫؍‬ ؊140 mV and E 2 (sq/ox) ‫؍‬ ؊219 mV, where hq is FAD hydroquinone and ox is oxidized FAD, establish that the sq is not thermodynamically stabilized (⌬E ‫؍‬ E 2 ؊ E 1 ‫؍‬ ؊79 mV). Results with N386D CPD-PL support our earlier hypothesis of a kinetic barrier to sq oxidation associated with proton transfer. Both E 1 and E 2 are upshifted by ϳ100 mV in this mutant; replacing the N5-proximal Asn with Asp decreases the driving force for sq oxidation. However, this Asp alleviates the kinetic barrier, presumably by acting as a proton shuttle, because the sq in N386D CPD-PL oxidizes orders of magnitude more rapidly than wild type. These data clearly reveal, as suggested for plant CRYs, that an N5-proximal Asp can switch on proton transfer and modulate sq reactivity. However, the effect is context-dependent. More generally, we propose that PLs and CRYs tune the properties of their N5-proximal residue to adjust the extent of proton transfer, H-bonding patterns, and changes in protein conformation associated with electron transfer at the flavin. Electron transfer (ET)2 within and between proteins is a ubiquitous and essential molecular process in biology. Proteins must tightly control the rates of ET and lifetimes of radical intermediates to use this fundamental chemical reaction for a vast array of cellular functions. A significant control mechanism involves coupling of the ET to a proton transfer (PT) (1-3). The PT ensures heightened discrimination toward subtle changes in protein structure. As compared with ET, it has a much shorter range and is very sensitive to the relative orientation of a few atoms (the proton, donor, and acceptor), and its kinetics and thermodynamics can be adjusted dramatically by tuning pK a values over a very large range (3). Consequently, proton-coupled ET (PCET) is widespread in nature, notably in biological energy conversion and redox catalysis. Understanding the mechanisms for PCET and identifying its role in protein evolution are central problems in chemical biology.Flavoproteins are major players in cellular redox reactions (4). The flavin cofactor can exist in three redox states: the two-electron reduced hydroquinone (hq), which is often anionic; the one-electron reduced semiquinone (sq), as a neutral or anionic radical; and the fully oxidized form (ox) (Fig. 1a). Flavoprotein...

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Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

Damiani

Nostedt

O’Neill

2011

Journal of Biological Chemistry

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“…In the neutral radical (FADH • ) and the anionic fully reduced (FADH − ) states of the FAD cofactor in prototypical photolyases, the N5 atom of the isoalloxazine moiety is protonated and hydrogen bonded to an Asn residue (17,27). Apparently, this stabilizing function is taken over by a conserved water molecule in PhrB that is held in place by additional hydrogen bonds with the side chain of Arg369 and the backbone carbonyl group of Tyr391 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

Zhang

Scheerer

Oberpichler

et al. 2013

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

178

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The (6-4) photolyases use blue light to reverse UV-induced (6-4) photoproducts in DNA. This (6-4) photorepair was thought to be restricted to eukaryotes. Here we report a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens, and propose that (6-4) photolyases are broadly distributed in prokaryotes. The crystal structure of photolyase related protein B (PhrB) at 1.45 Å resolution suggests a DNA binding mode different from that of the eukaryotic counterparts. A His-His-X-X-Arg motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. The PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore and a [4Fe-4S] cluster bound to the catalytic domain. A significant part of the Fe-S fold strikingly resembles that of the large subunit of eukaryotic and archaeal primases, suggesting that the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. Our study presents a unique prokaryotic (6-4) photolyase and proposes that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family.DNA repair | UV light | energy transfer

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“…On the flavin cofactor side, N378 is opposite to the N5 position of flavin, and its side carbonyl group forms a hydrogen bond with the N(5)H group of FADH or FADH À . Destroying the interaction would abolish the ability of photolyase to stabilize the FADH radical 16 . These five critical residues at the active site that were studied are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

The molecular origin of high DNA-repair efficiency by photolyase

Tan

Liu

et al. 2015

Nat Commun

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The primary dynamics in photomachinery such as charge separation in photosynthesis and bond isomerization in sensory photoreceptor are typically ultrafast to accelerate functional dynamics and avoid energy dissipation. The same is also true for the DNA repair enzyme, photolyase. However, it is not known how the photoinduced step is optimized in photolyase to attain maximum efficiency. Here, we analyse the primary reaction steps of repair of ultraviolet-damaged DNA by photolyase using femtosecond spectroscopy. With systematic mutations of the amino acids involved in binding of the flavin cofactor and the cyclobutane pyrimidine dimer substrate, we report our direct deconvolution of the catalytic dynamics with three electron-transfer and two bond-breaking elementary steps and thus the fine tuning of the biological repair function for optimal efficiency. We found that the maximum repair efficiency is not enhanced by the ultrafast photoinduced process but achieved by the synergistic optimization of all steps in the complex repair reaction.

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Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair

Cited by 30 publications

References 38 publications

Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

The molecular origin of high DNA-repair efficiency by photolyase

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