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Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB.

ABSTRACT: Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N(6)-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher k(cat) and K(m) for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this "k(cat)/K(m) compensation," which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O(2) substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O(2) binding before nucleotide substrate binding.

SUBMITTER: Yu B

PROVIDER: S-EPMC2725012 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:E. coli AlkB and human ALKBH2 belong to the AlkB family enzymes, which contain several α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases that repair alkylated DNA. Specifically, the AlkB enzymes catalyze decarboxylation of α-KG to generate a high-valent Fe(IV)-oxo species that oxidizes alkyl groups on DNA adducts. AlkB and ALKBH2 have been reported to differentially repair select etheno adducts, with preferences for 1,N6-ethenoadenine (1,N6-εA) and 3,N4-ethenocytosine (3,N4-εC) over 1,N2-ethenoguanine (1,N2-εG). However, N2,3-ethenoguanine (N2,3-εG), the most common etheno adduct, is not repaired by the AlkB enzymes. Unfortunately, a structural understanding of the differential activity of E. coli AlkB and human ALKBH2 is lacking due to challenges acquiring atomistic details for a range of substrates using experiments. This study uses both molecular dynamics (MD) simulations and ONIOM(QM:MM) calculations to determine how the active site changes upon binding each etheno adduct and characterizes the corresponding catalytic impacts. Our data reveal that the preferred etheno substrates (1,N6-εA and 3,N4-εC) form favorable interactions with catalytic residues that situate the lesion near the Fe(IV)-oxo species and permit efficient oxidation. In contrast, although the damage remains correctly aligned with respect to the Fe(IV)-oxo moiety, repair of 1,N2-εG is mitigated by increased solvation of the active site and a larger distance between Fe(IV)-oxo and the aberrant carbons. Binding of non-substrate N2,3-εG in the active site disrupts key DNA-enzyme interactions, and positions the aberrant carbon atoms even further from the Fe(IV)-oxo species, leading to prohibitively high barriers for oxidative catalysis. Overall, our calculations provide the first structural insight required to rationalize the experimentally-reported substrate specificities of AlkB and ALKBH2 and thereby highlight the roles of several active site residues in the repair of etheno adducts that directly correlates with available experimental data. These proposed catalytic strategies can likely be generalized to other α-KG/Fe(II)-dependent dioxygenases that play similar critical biological roles, including epigenetic and post-translational regulation.

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Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB.

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