Catalytic Mechanism of the Bacterial Non-Heme Fe(II) and 2-Oxoglutarate Dependent Enzyme AlkB with Single-Stranded DNA Containing Complex Guanine Adducts

Abstract

The bacterial nonheme Fe(II)/2-oxoglutarate (2OG)-dependent enzyme AlkB repairs alkylation damages in single-stranded DNA (ss-DNA) nucleotide bases. This study examines for the first time the reaction mechanism of the AlkB-catalyzed repair of alkylated and exocyclic guanine adducts (GAs) in single-stranded DNA induced by everyday chemical exposures associated with cancers and other genetic disorders. The studied substrates include N2-furfurylguanine (FF-dG), N2-tetrahydrofuran-2-yl-methylguanine (HF-dG), 3-(2'-deoxy-β-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-α]purin-10(3H)-one (α-OH-PdG), 3-(2'-deoxy-β-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-α]purin-10(3H)-one (γ-OH-PdG), and 3-(2'-deoxy-β-D-erythro-pentofuranosyl) pyrimido[1,2-α]purin-10(3H)-one (M₁dG). Using molecular dynamics-based combined quantum mechanics/molecular mechanics (QM/MM) and QM calculations, we provide unique mechanistic insights into AlkB's catalytic reaction pathways with ss-DNA containing complex alkylated/exocyclic GAs in strong correlation to experimental studies. While HF-dG, FF-dG, α-OH-PdG, and γ-OH-PdG are repaired through C-H hydroxylation, M₁dG follows epoxidation. The study elucidated that the repair mechanism favors the open tautomer of γ-OH-PdG and the closed tautomer of α-OH-PdG, respectively, in agreement with experimental studies, due to the preferable SCS interactions and the catalytic domain's loop L1 and L4 dynamics. Our study also elucidated that the posthydroxylation/postepoxidation steps proceed in water rather than the enzyme. The results reveal the unique catalytic mechanism of AlkB with ss-DNA containing complex GAs, which can be used in drug design and metalloenzyme redesign.

Figure 1.

The crystal structure of the AlkB complexed with m1G substrate (PDB ID: 3KHC) features the C-terminal extension (CTE) in sky blue, the N-terminal extension (NTE) in cornflower blue, the ss-DNA in corol, active site residues in yellow, and the nucleotide recognition lid in light pink.

Figure 2.

The general catalytic mechanism of non-heme Fe(II)/2OG enzymes is shown, with the studied reactions highlighted in the dotted box.

Figure 3.

AlkB substrates studied in this work. a) α-OH-PdG, b) γ-OH-PdG, c) FF-dG, d) HF-dG, e) M₁dG.

Figure 4.

The proposed mechanism for the repair of the complex alkylated – a) HF-dG b) FF-dG and exocyclic adducts – c) α-OH-PdG, d) γ-OH-PdG e) M₁dG by AlkB.

Figure 5.

Residues forming the hydrophobic pocket (shown in green) surrounding the overlaid substrates in AlkB-Fe(IV)=O-GA complex. Substrates are colored as follows:

Figure 6.

The SCS interactions in a) HF-dG b) FF-dG c) CγG d) OγG substrates from 1μs MD simulation of the AlkB- ferryl complex. The frames are selected based on the average structure from the equilibrated portion of the MD simulations and capture the representative interactions observed throughout the simulation.

Figure 7.

The SCS interactions in a) CαG, b) OαG, c) M₁dG substrates from 1μs MD simulation of the AlkB-ferryl complex.The frames are selected based on the average structure from the equilibrated portion of the MD simulations and capture the representative interactions observed throughout the simulation.

Figure 8.

Overlaid average MD structure of AlkB-Fe(IV)=O-GA complexes for each substrate with the flexible loop labeled. The AlkB ferryl complex are colored as follows: .

Figure 9.

Analysis of FF-dG and HF-dG substrates. (a,c) QM/MM energy profile at the QM(B3)/MM B3LYP level of theory (in kcal/mol) for the HAT and rebound hydroxylation steps of FF-dG and HF-dG, respectively. (b,d) Geometries of the stationary points in the HAT and rebound steps of FF-dG and HF-dG, respectively, with structural parameters shown in black (distance in Å, angle in degree) and spin population shown in blue.

Figure 10.

Analysis of OγG and CαG substrates. (a,c) QM/MM energy profile at the QM(B3)/MM B3LYP level of theory (in kcal/mol) of the HAT and rebound hydroxylation steps of OγG and CαG, respectively. (b,d) Geometries of the stationary points in the HAT and rebound steps of OγG and CαG, respectively, with structural parameters shown in black(distance in Å, angle in degree) and spin population shown in blue.

Figure 11.

The relative reaction profiles at the QM(B3)/MM B3LYP level of theory of the substrates repaired by AlkB enzyme. Each of the reaction paths starts on its on RC1 and the energies (in kcal/mol) of all RC1s are set to zero.

Figure 12.

a) The electron transfer pathway involved in the HAT by ferryl complex. b) Spin natural orbitals (SNOs) and their occupancies (in bracket) in the HAT TSγO1 for AlkB-OγG complex.

Figure 13.

Analysis of M₁dG substrate. a) QM/MM energy profile at the QM(B3)/MM B3LYP level of theory(in kcal/mol) of the epoxidation step. b) Geometries of the stationary points in the epoxidation step with structural parameters shown in black (distance in Å, angle in degree) and spin population shown in blue. c) The spin and the NBO values of the TSM₁1 and IMM₁1 structures of the M₁dG substrate prior to the epoxidation.