Vanderbilt Institute of Chemical Biology



Discovery at the VICB







A Closer Look into the Mechanisms of DNA Repair


By: Carol A. Rouzer, VICB Communications
Published:  September 19, 2016


Structural and computational studies of the DNA glycosylase AlkD reveal key interactions between the substrate and the protein's active site.  



DNA glycosylases are enzymes that remove damaged bases from DNA in the first step of the base excision repair pathway of DNA repair. Most of these enzymes work by flipping the damaged base out of its place in the double helix in order to transfer it into the enzyme's active site where cleavage of the glycosidic bond between the base and the deoxyribose will occur (see Figure 1, top). In turn, the enzyme temporarily fills the space vacated by the flipped base by inserting one or more amino acids into the helix. Recently, however, Vanderbilt Institute of Chemical Biology member Brandt Eichman and his laboratory showed that AlkD, a DNA glycosylase from Bacillus cereus that specifically targets positively charged lesions, does not use the base-flipping mechanism for catalysis [E. A. Mullins, et al., (2015) Nature, 527, 254]. Instead, the damaged base retains its position in the double helix (Figure 1, bottom) while three AlkD residues (Asp113, Trp109, and Trp187) cradle the deoxyribose ring. These studies suggested that C-H/π interactions between the tryptophan residues and the deoxyribose could play a role in catalysis. However, few examples of a catalytic function for C-H/π interactions exist. This led the Eichman lab to further explore the catalytic mechanism of AlkD (Z. D. Parsons, et al. J. Am. Chem. Soc., 138, 11485).



. (Top) Diagrammatic representation of the crystal structure of human alkyladenine DNA glycosylase (a base-flipping DNA glycosylase) in complex with a substrate. The damaged nucleobase (magenta) is no longer paired to its complement (green). Instead, it has flipped out of its position in the DNA duplex and occupies the AAG active site. An intercalating residue from the enzyme (gray) fills the space left by the damaged base. (Bottom) Diagrammatic representation of the crystal structure of Bacillus cereus AlkD in complex with a substrate. Note that the damaged nucleobase (magenta) and its complement (green) remain stacked in the DNA duplex. Figure kindly provided by Brandt Eichman (Copyright 2015, Brandt Eichman).


In their initial studies, the researchers had focused on the interaction between AlkD and DNA containing a 3-methyl-2′-deoxyadenosine (d3mA) lesion (Figure 2). In this case, the methyl group of the damaged base is found in the minor groove of the DNA. To ascertain if their findings regarding the mechanism of AlkD also applied to major groove lesions, the investigators chose to study the interaction of the enzyme with DNA containing 7-methyl-2′-deoxyguanosine (d7mG) (Figure 2). They began their experiments by obtaining the crystal structure of a complex of AlkD, 7-methyl-guanine (7mG), and DNA containing 1-aza-2′,4′dideoxyribose (1aR). This complex mimics the structure of the DNA strand following cleavage of the glycosidic bond that leads to the formation of an oxocarbenium ion on the deoxyribose moiety (Figure 2). The crystal structure (Figure 3) was very similar to that obtained previously for a complex containing 3mA instead of 7mG. The damaged base and its opposing dC remained in place within the DNA double helix. Trp109 and Trp187 were closely associated with the 1aR moiety, which substituted for the deoxyribose oxocarbenium ion, and Asp113 was hydrogen-bonded to a water molecule, placing it in position to attack the positively charged nitrogen atom of 1aR.




FIGURE 2. Structures of d3mA and d7mG within a DNA chain (top) and the corresponding free bases along with 1aR within a DNA chain.




FIGURE 3. (A) Structure of the AlkD-d7mG-DNA complex The protein is shown in green, the free base lesion (labeled 7mGua in the figure) is purple, and the complementary dC is yellow. (B) Close-up view of the enzyme active site, highlighting 1aR in close association with Trp109 and Trp187. Asp113 hydrogen-bonds to a water molecule that is, in turn, hydrogen-bonded to the nitrogen atom in the ring of 1aR that serves as an analog for the carbenium ion intermediate during catalysis. Figure reproduced by permission from Z. D. Parsons, et al. J. Am. Chem. Soc., 138, 11485. Copyright 2016 American Chemical Society.



The crystal structure results strongly supported the hypothesis that C-H/π interactions play an important role during AlkD-mediated catalysis. To further test this hypothesis, the investigators constructed site-directed mutants of AlkD, replacing Asp113, Trp109, and Trp187 each with alanine. Michaelis-Menten kinetic analyses of the activities of the three mutant enzymes revealed that the alterations had little to no effect on substrate binding (KM), but all of them reduced the rate of catalysis (kcat) by over 99%. These findings confirmed that all three residues are critical to AlkD's activity and suggested that C-H/π interactions mediated by the two tryptophan residues were as important as the electrostatic and hydrogen-bonding role played by Asp113, a somewhat surprising finding.


To further understand the role of C-H/π interactions in AlkD catalysis, the researchers created a computational model of the enzyme's active site, including the three critical amino acid residues, and the d7mG nucleoside. They showed that this model predicted an activation energy for glycosidic bond cleavage that was in good agreement with experimental values. They then substituted each of the three amino acids with "ghost atoms" and calculated the effects of the changes on reaction energetics while maintaining all of the other components in a fixed position. This model design facilitated the detection of changes in electrostatic effects but not conformational effects due to the inability of the various components to alter position. The model predicted that removal of Asp113 would cause a 99.7% reduction in catalytic activity, in good agreement with the 99.4% reduction actually observed. This result was consistent with the model's ability to detect changes in the primarily electrostatic effects of Asp113. The model's prediction that removal of Trp187 would lead to an 88% loss of activity was in reasonable agreement with the measured value of 99.4%, suggesting that Trp187's role might be primarily electrostatic in nature. The close proximity of Trp187 to the oxocarbenium ion and its ability to donate electron density from its π-system further supported this conclusion. In contrast, the model predicted that removal of Trp109 would actually increase activity rather than decrease it by over 99% as was experimentally observed. This result suggests that Trp109's role might be primarily to stabilize a change in conformation that favors glycosidic bond cleavage.


Together the data confirm the prior findings that AlkD functions by a mechanism that is distinct from that of most DNA glycosylases. Base flipping does not occur, and both Trp109 and Trp187 contribute significantly to catalysis via C-H/π interactions (Figure 4). The modeling data suggest that these interactions stabilize the transition state of the reaction by both contributing negative charge to the oxocarbenium ion intermediate and by promoting a conformation that facilitates glycosidic bond cleavage. These findings provide important new insight into the mechanism of an interesting DNA glycosylase and establish a role for C-H/π interactions in the catalytic mechanism of the enzyme. The authors point out that the deoxyribose ring of DNA is particularly well suited to engage in C-H/π interactions with π-rich systems such as are found in tryptophan residues, and that such interactions are abundant in DNA binding proteins. Therefore, it is likely that further study will demonstrate the importance of C-H/π interactions in the function of many DNA metabolizing enzymes.



FIGURE 4. Proposed mechanism for glycosidic bond cleavage by AlkD. The positive charge on N7 of d7mG naturally destabilizes the glycosidic bond, facilitating cleavage. The enzyme promotes the reaction by stabilizing the carbenium ion intermediate through electrostatic interactions with Asp113, and possibly also via CH/π interactions with Trp187 and Trp109.  The Trp residues may also stabilize the intermediate conformationally. In the final step, a water molecule, guided by interactions with Asp113 adds to the carbenium ion to generate the deoxyribose product. Figure reproduced by permission from Z. D. Parsons, et al. J. Am. Chem. Soc., 138, 11485. Copyright 2016 American Chemical Society.




ViewJACS article: A Catalytic Role for C–H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD







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