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Biophys Physicobiol
2015 Dec 22;12:139-44. doi: 10.2142/biophysico.12.0_139.
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Structural role of two histidines in the (6-4) photolyase reaction.
Yamada D
,
Iwata T
,
Yamamoto J
,
Hitomi K
,
Todo T
,
Iwai S
,
Getzoff ED
,
Kandori H
.
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Photolyases (PHRs) are DNA repair enzymes that revert UV-induced photoproducts, either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts (PPs), into normal bases to maintain genetic integrity. (6-4) PHR must catalyze not only covalent bond cleavage, but also hydroxyl or amino group transfer, yielding a more complex mechanism than that postulated for CPDPHR. Previous mutation analysis revealed the importance of two histidines in the active center, H354 and H358 for Xenopus (6-4) PHR, whose mutations significantly lowered the enzymatic activity. Based upon highly sensitive FTIR analysis of the repair function, here we report that both H354A and H358A mutants of Xenopus (6-4) PHR still maintain their repair activity, although the efficiency is much lower than that of the wild type. Similar difference FTIR spectra between the wild type and mutant proteins suggest a common mechanism of repair in which (6-4) PP binds to the active center of each mutant, and is released after repair, as occurs in the wild type. Similar FTIR spectra also suggest that a decrease in volume by the H-to-A mutation is possibly compensated by the addition of water molecule( s). Such a modified environment is sufficient for the repair function that is probably controlled by proton-coupled electron transfer between the enzyme and substrate. On the other hand, two histidines must work in a concerted manner in the active center of the wild-type enzyme, which significantly raises the repair efficiency.
Figure 1. (a) Molecular structures of two thymines (center) and UV-induced photoproducts, CPD (left) and (6-4) PP (right). (b) The (6-4) PP binding site of (6-4) PHR (structure from PDB: 3cvu). FAD chromophore, (6-4) PP and residues are shown as a stick drawing using yellow, orange and green, respectively. The hydrogen-network is shown as a dotted line. The number of amino acid residues corresponds to Xenopus (6-4) PHR.
Figure 2. (a) The photorepair difference FTIR spectra of Xenopus (6-4) PHR; His-tagged sample in this study (black line) and GST-tagged sample in a previous study (purple line). The purple spectrum is reproduced from Zhang et al. [16]. (b) Black and red lines represent the spectra with the wild type and the H354A mutant of (6-4) PHR under 2-min illumination, respectively. (c) Black and blue lines represent the spectra with the wild type and the H358A mutant of (6-4) PHR under 2-min illumination, respectively. One division of the y-axis corresponds to 0.008 absorbance units.
Figure 3. Comparison of the light-induced difference FTIR spectra of Xenopus (6-4) PHR for the wild type (black line) under 2-min illumination, the H358A mutant (blue line) under 10-min illumination (a) and the H358A mutant spectrum (8-fold expansion of the blue spectrum in (a) (green line) (b). One division of the y-axis corresponds to 0.01 absorbance units.
Figure 4. Comparison of the light-induced difference FTIR spectra of Xenopus (6-4) PHR for the wild type (black line) under 2-min illumination, the H354A mutant (red line) under 30-min illumination (a) and the H354A mutant spectrum (10-fold expansion of the blue spectrum in (a) (green line) (b). One division of the y-axis corresponds to 0.01 absorbance units.
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