Modulating Enzyme's Conformational Space: Impact of Substrate Binding, Mode Alteration, and Active Site Mutation in DapC, an Aminotransferase Enzyme of Lysine Biosynthetic Pathway.

Abstract

The microbial aminotransferase enzyme DapC is vital for lysine biosynthesis in various Gram-positive bacteria, including Mycobacterium tuberculosis. Characterization of the enzyme's conformational dynamics and identifying the key residues for ligand binding are crucial for the development of effective antimicrobials. This study employs atomistic simulations to explore and categorize the dynamics of DapC in comparison to other classes of aminotransferase. DapC undergoes an open-to-closed conformational change upon substrate binding, characterized by the movement of the N-terminal α2 helix, akin to that observed in the class Ib aspartate aminotransferase from Thermus thermophilus. Based on sequence similarity, essential dynamics, and the absence of the characteristic hinge movement, DapC is classified as a class Ib aminotransferase of type-I pyridoxal-5'-phosphate (PLP)-dependent enzyme. In the open state of DapC, two binding modes of glutamate, namely, canonical and alternate, separated by a dihedral rotation, are equally preferred. The closed state prefers the canonical binding mode, which is favorable for catalysis. In the case where the substrate binds in the alternate mode, a low-barrier dihedral rotation generates the canonical mode for efficient catalysis. The presence of two highly conserved residues, Phe14 and Gln31, stabilizes the closed state of substrate-bound DapC. Mutations of these residues disrupt the crucial hydrophobic interactions with the substrate, causing the enzyme to shift to an open state. While Phe14 has a dominant role, Gln31 is less consequential in regulating the conformational change, while the double mutation leads to a rapid conformation change.