DOI: 10.1073/pnas.0812938106. Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB

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

10.1073/pnas.0812938106

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

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.

Bibliography

Yu, B., & Hunt, J. F. (2009). Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB. Proceedings of the National Academy of Sciences, 106(34), 14315â14320.

Authors 2

Bomina Yu (first)
John F. Hunt (additional)

References 27 Referenced 84

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Dates

Type	When
Created	16 years ago (Aug. 12, 2009, 12:25 a.m.)
Deposited	3 years, 3 months ago (May 23, 2022, 5:13 p.m.)
Indexed	6 days, 18 hours ago (Aug. 27, 2025, 11:56 a.m.)
Issued	16 years ago (Aug. 25, 2009)
Published	16 years ago (Aug. 25, 2009)
Published Online	16 years ago (Aug. 25, 2009)
Published Print	16 years ago (Aug. 25, 2009)

Funders 0

None

BibTeX

@article{Yu_2009, title={Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB}, volume={106}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0812938106}, DOI={10.1073/pnas.0812938106}, number={34}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Yu, Bomina and Hunt, John F.}, year={2009}, month=aug, pages={14315–14320} }

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