Authors: Evans Boateng-Boakye | Shubham Chatterjee | Xianhao Zhou | Jose P. Madriaga | Jian Ma | Samuel Haworth | Yi-Tzai Chen | Zhiyuan Peng | Bongsup P. Cho | G Andrés Cisneros | Deyu Li
Source: ChemRxiv (article, 2026-03-04)
Field: Chemistry — Inorganic Chemistry

Comprehensive Kinetic and Mechanistic Analysis of AlkB-Catalyzed Oxidation of Alkyl Modifications Despite extensive studies of the 2-oxoglutarate/Fe(II)-dependent dioxygenase AlkB, the determinants of its substrate selectivity and specificity remain incompletely defined. Reported kinetic parameters vary widely across studies due to differences in nucleic acid sequence and context, reaction conditions, analytical approaches, and limited substrate scope, complicating cross-study comparisons and obscuring the enzyme’s catalytic capacity. In particular, AlkB’s ability to process exocyclic dimethyl modifications has not been systematically evaluated within a unified kinetic framework alongside other alkyl substrates. Here, we present a comprehensive kinetic analysis of wild-type E. coli AlkB using a chemically diverse panel of alkyl modifications inserted at a defined position within DNA, all examined under identical conditions. The results provide the most comprehensive and quantitative kinetic study of AlkB on thirteen substrates in both single-stranded and double-stranded DNA. These results reveal that exocyclic dimethyl modifications are demethylated more efficiently than their corresponding monomethyl counterparts and that it proceeds through a stepwise, biphasic mechanism with a kinetically favored first demethylation step. Within this framework, we identify N4,N4-dimethylcytidine (m44C) and N2,N2-dimethylguanine (m22G) as a previously unreported wild-type AlkB substrate together with a known substrate, N6,N6-dimethyladenine (m66A). We further show that AlkB demethylation is nucleobase position-, substrate-, and strand-context dependent. Molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) studies to investigate AlkB catalysis of m22G and m2G as a representative dimethyl modification and its monomethyl counterpart show structural and dynamical differences depending on the substrate, giving rise to differences in interaction of the substrate and the percentage of catalytically competent structures. The calculated reaction energy profiles show that although hydrogen atom transfer (HAT) kinetics in the slowest step are comparable for both substrates, the m22G is thermodynamically driven, with the intermediate being significantly more stabilized. We also demonstrated that AlkB has activity towards alkyl modification in RNA. Together, this work reconciles discrepancies in the AlkB literature, establishes a unified kinetic and mechanistic framework for exploring the demethylation of analogous AlkB homologs’ diverse substrates, including dimethyl modifications, and refines our understanding of how AlkB and its homologs handle chemically and sterically diverse alkyl modifications. AlkB ; Dioxygenase ; Substrate (aquarium) ; Alkyl ; Nucleobase ; Demethylation ; Kinetic energy ; Catalysis ; Reaction mechanism Metal-Catalyzed Oxygenation Mechanisms ; Epigenetics and DNA Methylation ; Microbial metabolism and enzyme function