The Catalytic Efficiency and Conformational Dynamics of Escherichia coli DNA Repair Enzyme AlkB

Enzymes catalyze specific reactions in almost all cellular processes, including DNA replication and repair, transcription, translation, signal transduction and energy production. Therefore, extensive efforts are underway to understand the functions and mechanisms of these processes. The potential contribution of the conformational dynamics of enzymes to their high catalytic power has received particular attention in the last decades. Studies indicate that protein dynamics are involved in substrate binding and product release; however, the role of dynamics in catalysis is still controversial. Here, we investigate the substrate-dependent dynamic properties of the Escherichia coli AlkB protein, and the role of a specific dynamic transition in the efficiency of the catalytic reaction cycle. AlkB is an iron/2-oxoglutarate (Fe(II)/2OG) dependent dioxygenase, which removes certain cytotoxic alkyl lesions from DNA and RNA bases that are not repaired by other known mechanisms. Using Fe(II) as a cofactor and 2OG and molecular oxygen as co-substrates, AlkB catalyzes a multistep redox reaction in which first, 2OG is oxidized yielding succinate, carbon dioxide and a reactive oxyferryl (Fe(IV)=O) intermediate; second, the alkylated base is hydroxylated by the Fe(IV)=O intermediate, and third, the hydroxylated base spontaneously resolves upon release from the enzyme. Our fluorescence and NMR spectroscopic data demonstrate that a microsecond-tomillisecond timescale conformational transition in the nucleotide recognition lid (NRL) of AlkB regulates the correct sequential order of substrate binding, i.e. Fe(II) and 2OG first, followed by the DNA substrate. By combining isothermal titration calorimetry with NMR, we show that less than 20% of the residues in AlkB become ordered during this conformational transition, indicating that this conformational change is mostly localized to the NRL, while the conformation of the dioxygenase core is minimally altered. In mutant AlkB variants that perturb the dynamics of this transition, 2OG is oxidized generating the Fe(IV)=O intermediate; however, the reaction cycle cannot be completed due to the premature release of the alkylated DNA substrate, leading to uncoupled turnover of 2OG. These data demonstrate that the conformational dynamics control the catalytic efficiency of AlkB. Our results further extend the view on the role of protein dynamics in substrate binding or product release by emphasizing the importance of protein dynamics for coupling sequential sub-reactions in a complex multistep reaction cycle. This finding illustrates a striking example of the relation between protein dynamics and overall enzyme efficiency.