Molecular Dynamics Simulations for the Michaelis Complex of Ectoine Synthase (EctC)

Abstract

Ectoine is a chemical chaperone synthesised and used by bacteria to defend against osmotic stress. Although it has already gained attention from the pharmaceutical and cosmetic industries, thanks to its hydrating and cell-protecting properties, the reaction mechanism of its final synthesis step is still not fully understood. The ultimate step of ectoine biosynthesis is catalysed by the ectoine synthase enzyme (EctC), which requires an iron ion for substrate binding and overall enzymatic activity. Even though a crystal structure for Paenibacillus lautus EctC—substrate complex is available (PDB: 5ONN), it is not very informative with respect to the geometry of the active site because: (1) the crystal was obtained at a pH value far from the enzyme’s pH optimum, (2) the electron density at the Fe position is weak, and (3) the Fe-ligand distances are too long. To fill this gap, in this work we have used classical molecular dynamics simulations to model the enzyme-substrate (N-gamma-acetyl-L-2,4-diaminobutyric acid) complex of Paenibacillus lautus EctC (PlEctC). Since PlEctC is a homodimeric protein, MD simulations were carried out for a dimer with various plausible occupancies by the substrate and for two plausible coordination geometries around the catalytic Fe ion: tetrahedral and octahedral. MD results revealed that the presence of the ligand has a stabilising effect on the protein structure, most notably on a short helix 112–118, which flanks the entrance to the active site. The most important amino acids for substrate binding are Trp21, Arg25, Asn38, Thr40, and Tyr52, which were also identified in the crystal structure. Importantly, the substrate can easily adopt a conformation suitable for the progress of the catalytic reaction, and it does so spontaneously for the octahedral 6-coordinate geometry of the iron cofactor or with a low energy penalty (ca. 3 kcal/mol) in the case of 4-coordinate tetrahedral geometry. Simulations for different substrate occupancy states did not reveal any signs of cooperativity between the two monomers.