Journal of the American Chemical Society Figure 4. Percentages (y-axis) of the four species in the AlkB reactions on EA at 1, 2, 5, 10, 20, 40, 60, and 80 min incubation time points (x- axis) (see data in Table S1, SI ). The combined percentages of the four species are 100%. of the adenine base. The mono-oxidized intermediates 1 and 2 (Figure 1a) equilibrate between the ring-closed and ring- opened forms, both bearing an observed m/z of 1231.66. The opened-ring intermediate 2 can take on a conformation in which it can form a Watson−Crick base pair and thereby not block DNA replication. This hypothesis is strongly supported by in vivo experiments showing EA is neither toxic nor mutagenic in AlkB-proﬁcient E. coli cells.6 In the current study, we observed EA to be completely repaired to the adenine base, 6 which requires oxidation reactions at both the N1 and N - attached carbon atoms (Figures 1a and 3b). A clue as to the reaction path of EA was the observation of an AlkB-induced glycol (m/z of 1235.67) identical to the glycol formed during removal of the etheno bridge from eA (Figure 1d). In order for EA to form a glycol (intermediate 4, Figure 1a) such as that of eA, it would need to experience two oxidation reactions (Figure 1a). The presence of the glycol and the observation of other intermediates indicated by their masses in Figure 1a allowed us to assemble a reaction network that would explain the removal of the ethano-bridge from EA. As shown in Figure 1a, AlkB can oxidize each carbon of the saturated two carbon bridge and completely restore the undamaged adenine base. The ﬁrst oxidation can occur either at the N1- or N6-attached carbon of the EA moiety and generate intermediate 1 or 3, respectively. Each ring-closed intermediate will equilibrate with its ring- opened form 2 or 7. Further oxidation of 1 or 3 will generate a common intermediate 4, which can also exist in the respective ring-opened form of 5 or 8. Further oxidation of the ring- opened intermediate 2 or 7 will also generate 5 or 8. The two- carbon unit attached to EA in intermediate 5 or 8 can then be released as glyoxal to form the undamaged adenine (Figure 1a), identical to the ﬁnal step of eA repair after epoxide hydrolysis (Figure 1d).10 If the glycol 4 or the α-hydroxy aldehyde intermediate 5 or 8 is further oxidized, two aldehyde products will form: the terminal carbon will be released as formaldehyde, and the other carbon attached to either the N6- or N1-position of the adenine base will generate intermediates 6 or 9. The formation of intermediate 6 or 9 should adopt a mechanism similar to typical oxidative cleavage of a glycol to two aldehydes by sodium periodate.24,25 It is possible that intermediate 6 or 9 can be spontaneously converted to the adenine base by amide hydrolysis, or they may remain side products in the reaction rather than formal intermediates. To elucidate the kinetic distributions of the four new species in the AlkB reaction, we carried out a series of enzymatic reactions at 37 °C with varied time durations at a ﬁxed enzyme−substrate ratio. We chose the time points at 1, 2, 5, 10, Article 20, 40, 60, and 80 min to quench the reactions and subsequently carried out HPLC-ESI-TOF analysis using an autosampler set to 5 °C in an eﬀort to keep the products and intermediates intact. As presented in Figure 4, we observed that the EA starting material was completely consumed even after the 1 min incubation. This result indicates that the AlkB reaction on EA happened very fast under the current reaction conditions. We also observed that a major species in all mass spectra was the peak at m/z of 1231.66 (from 77.4% to 91.5%, Figure 4 and Table S1, SI), which matches up with intermediate with one extra oxygen atom added to EA. The percentage of this mono-oxidation intermediate decreased as incubation at 37 °C went to longer times. The intermediate with two extra oxygen atoms added to EA displayed a relative low percentage for all time periods. The ratios of the fully repaired product (undamaged adenine) and the EA+2 species (intermediate 6 or 9 in Figure 1a) gradually increased as the incubation time increased (Figure 4). The ratio of adenine increased from 2.6% to 10.0% during the 1−80 min interval. By performing the HPLC-ESI-TOF analysis of the incubation of EA with AlkB using an autosampler at room temperature, we observed a signiﬁcantly increased amount of adenine (Figure 3b), which might be generated from the spontaneous decomposition of the oxidized intermediates. AlkB Demethylates m6A. The ability of AlkB to demethylate an m6A-containing oligonucleotide was addressed. The observed m/z of 1224.73 for the −4 charge monoisotopic peak from the m6A starting material was seen in the absence of AlkB (Figure 3c). In the presence of the AlkB protein, three peak envelopes are seen in the mass spectrum (Figure 3d). The peak at 1224.73 corresponds to the unreacted 16mer containing m6A (about 68% for a 60 min incubation). The 1221.24 peak envelope is assigned to the unmodiﬁed base adenine (15%). The peak envelope at 1228.72 (17%) matches the theoretical MW of the HO-m6A very well (1228.71 at −4 charge state). It decomposes to A as the reaction time progresses (Figure S3, SI). We believe this to be the ﬁrst instance of observing the hydroxymethyl intermediate in the AlkB reaction for simple methyl adducts, which strongly supports AlkB’s oxidative demethylation mechanism. With respect to the 100% conversion of m1A under similar reaction conditions, the incomplete repair and buildup of hydroxy intermediate seen for the AlkB reaction with m6A suggest that m6A is more sluggishly processed than m1A. Unlike the AlkB reaction on m6A, we did not observe a hydroxy intermediate (i.e., no HO-m1A) in the m1A reaction. The diﬀerent behaviors of m1A and m6A could originate from the charge state of the lesions: at physiological pH, m1A is positively charged, whereas m6A is neutral. The positively charged HO-m1A intermediate should be less stable than the neutral HO-m6A, leading to faster accumulation of adenine and inability to observe the hydroxy intermediate. ■ DISCUSSION Biological Implications of AlkB Repair of EA. The alleviation of EA toxicity by AlkB in E. coli provides an opportunity to investigate further the repair eﬃciency of such lesions by human homologues, such as hABH2. If hABH2 or its homologues can eﬃciently repair EA in vivo, the anticancer eﬃcacy of BCNU (and possibly other DNA damaging anticancer drugs) would be greatly reduced. The design of a chemical inhibitor to block the repair activity of AlkB or hABH2 on EA should enhance the anticancer eﬃcacy of 8899 dx.doi.org/10.1021/ja3010094 | J. Am. Chem. Soc. 2012, 134, 8896−8901