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

Department of Biological Sciences, 702A Fairchild Center, MC2434, Columbia University, New York, NY 10027, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2009; 106(34):14315-20. DOI: 10.1073/pnas.0812938106
Source: PubMed


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.

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    • "The relationship between initial velocity, v, and ␧A concentration, [␧A], conformed to a Michaelis–Menten equation (Fig. 3B), as indicated by linearity in a Hanes–Woolf plot of [␧A]/v versus [␧A] (Fig. 3C). It has been reported previously that compared to 1-meA or 3-meC adducts, ␧-DNA substrate was repaired much less efficiently by AlkB, with approximate K M and k cat values of 59.7 ± 14.2 ␮M and 0.13 ± 0.05 min −1 , respectively [5]. Our analysis using AlkB and 40-mer ␧A as substrate yielded K M and k cat to be 67.4 ␮M and 0.134 min −1 , respectively. "
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    • "Structural disorder predictions were performed with the VSL1 algorithm [47] and DISOPRED2 [48]. The structural model of the Ofd2 core domain was derived from an E. coli AlkB template from Yu and Hunt [32] (Protein Databank identifier 3I3Q) and the illustration was generated with PyMOL [49]. "
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    ABSTRACT: 2-Oxoglutarate (2OG) dependent dioxygenases are ubiquitous iron containing enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. They participate in a wide range of biological processes including collagen biosynthesis, fatty acid metabolism, hypoxic sensing and demethylation of nucleic acids and histones. Although substantial progress has been made in elucidating their function, the role of many 2OG dioxygenases remains enigmatic. Here we have studied the 2OG and iron (Fe(II)) dependent dioxygenase Ofd2 in Schizosaccharomyces pombe, a member of the AlkB subfamily of dioxygenases. We show that decarboxylation of 2OG by recombinant Ofd2 is dependent on Fe(II) and a histidine residue predicted to be involved in Fe(II) coordination. The decarboxylase activity of Ofd2 is stimulated by histones, and H2A has the strongest effect. Ofd2 interacts with all four core histones, however, only very weakly with H4. Our results define a new subclass of AlkB proteins interacting with histones, which also might comprise some of the human AlkB homologs with unknown function.
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