Abstract
The binding of (E)-2-(acetamidomethylene)succinate (E-2AMS) to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD), steered MD (SMD), and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMS's pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzyme's hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMS's pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism.
Highlights
Vitamin B6 belongs to pyridine derivate, which mainly refers to pyridoxine, pyridoxal, pyridoxamine and their phosphonate esterification form [1]
The important hydrogen bond (H-bond) interaction mentioned above appears in the docked poses of E-2AMS
The docked complex structures show that the amide oxygen atom of trans-form E-2AMS forms two Hbonds with the oxyanion-hole residues (Ile41 and Leu107) but the cis-form substrate does not have the similar H-bonds
Summary
Vitamin B6 belongs to pyridine derivate, which mainly refers to pyridoxine, pyridoxal, pyridoxamine and their phosphonate esterification form [1]. Vitamin B6 exists in the active forms of pyridoxal-6-phosphate (PLP). PLP plays a vital role as the cofactor of a large number of essential enzymes, which focus many important physiological processes, including macronutrient metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and gene expression [2,3]. In these processes, PLP covalently binds to substrate and stabilizes carbanion intermediates adjacent to an amino group [3]. The classic clinical syndrome for vitamin B6 deficiency is a seborrhoeic dermatitis-like eruption, atrophic glossitis with ulceration, angular cheilitis, conjunctibitis, intertrigo, and neurologic symptoms of somnolence, confusion, and neuropathy [4]
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