Electrochemical Characterization of Escherichia coli Adaptive Response Protein AidB

Michael Hamill,Cintyu Wong,Sean Elliott,Marco Jost,Catherine Drennan,Nicholas Bene

doi:10.3390/ijms131216899

Abstract

When exposed to known DNA-damaging alkylating agents, Escherichia coli cells increase production of four DNA repair enzymes: Ada, AlkA, AlkB, and AidB. The role of three enzymes (Ada, AlkA, and AlkB) in repairing DNA lesions has been well characterized, while the function of AidB is poorly understood. AidB has a distinct cofactor that is potentially related to the elusive role of AidB in adaptive response: a redox active flavin adenine dinucleotide (FAD). In this study, we report the thermodynamic redox properties of the AidB flavin for the first time, both for free protein and in the presence of potential substrates. We find that the midpoint reduction potential of the AidB flavin is within a biologically relevant window for redox chemistry at −181 mV, that AidB significantly stabilizes the flavin semiquinone, and that small molecule binding perturbs the observed reduction potential. Our electrochemical results combined with structural analysis allow for fresh comparisons between AidB and the homologous acyl-coenzyme A dehydrogenase (ACAD) family of enzymes. AidB exhibits several discrepancies from ACADs that suggest a novel catalytic mechanism distinct from that of the ACAD family enzymes.

Highlights

Escherichia coli adaptive response is a process initiated by the exposure of cells to low doses of DNA alkylating agents, which have mutagenic and cytotoxic effects on cells [1,2,3]
We investigated the option of redox modulations caused by potential AidB substrates such as isovaleryl-coenzyme A (IVCoA) or DNA, providing a comparison between AidB and the acyl-coenzyme A dehydrogenase (ACAD) family of enzymes that have well-characterized and distinctive redox properties
The absorbance maximum for the AidB anionic semiquinone is 365 nm, with an isosbestic point at 409 nm observed during the transition from the oxidized to the semiquinone species (Figure 1A)

Summary

Introduction

Escherichia coli adaptive response is a process initiated by the exposure of cells to low doses of DNA alkylating agents, which have mutagenic and cytotoxic effects on cells [1,2,3]. The adaptive response process refers to the up-regulation of four proteins, Ada, AlkA, AlkB, and AidB, that reduce the effects of alkylation on DNA [3,4]. Out of these four proteins, three have known roles in the adaptive response as enzymes that repair DNA lesions via a variety of mechanisms: Ada as a methyltransferase [5,6,7,8], AlkA as a glycosylase [9,10], and AlkB as a dioxygenase [11,12,13,14,15]. AidB is similar to members of the acyl-coenzyme A dehydrogenase (ACAD) enzyme family [16], which contain a redox active flavin adenine dinucleotide (FAD) cofactor that catalyzes the α,β-dehydrogenation of acyl-coenzyme A (CoA) substrates (Scheme 1). AidB contains a FAD cofactor and displays isovaleryl-coenzyme A dehydrogenase (IVD)

Results

Discussion

Conclusion