Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora

Abstract

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl β-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Keywords: EPS production regulation; Erwinia amylovora; amylovoran; exopolysaccharide; fire blight; in vitro assays; inhibition constant; molecular docking; protein tyrosine phosphatase; virtual screening.

Scheme 1

Proposed reaction mechanism of Protein Tyrosine Phosphatases (PTPs). Residues are labeled according to EaAmsI numbering [19,22].

Scheme 2

Workflow used in this work for the identification of new putative EaAmsI inhibitors.

Figure 1

Two-dimensional diagrams and names of the six selected molecules resulting from the virtual screening.

Figure 2

(A) Dose–response curves of EaAmsI activity in the presence of N7G (black circles) and N8G (black triangles). (B) Michaelis–Menten plot of EaAmsI at increasing concentrations of N7G (0, 4, 8, and 16 µM represented as black, blue, green, and red dots, respectively). The lines represent the results of the data-fitting procedure described in the experimental section.

Figure 3

(A) Cartoon and molecular surface representation of EaAmsI bound to N7G as a result of the DOCK6 molecular docking. EaAmsI cartoons are colored from blue to red going from the N- to the C-terminal. Residues involved in the reaction or in the binding of N7G are sticks, while N7G is in ball-and-stick (carbon, light orange; oxygen, red). (B) Detail of N7G binding pose in the EaAmsI binding site. (C) Scheme of the interactions between the docked N7G molecule and EaAmsI. The scheme represents H-bonds (dashed lines) and van der Waals interactions (red spiked arcs).