Characterization and 1.57 Å resolution structure of the key fire blight phosphatase AmsI from Erwinia amylovora

Abstract

AmsI is a low-molecular-weight protein tyrosine phosphatase that regulates the production of amylovoran in the Gram-negative bacterium Erwinia amylovora, a specific pathogen of rosaceous plants such as apple, pear and quince. Amylovoran is an exopolysaccharide that is necessary for successful infection. In order to shed light on AmsI, its structure was solved at 1.57 Å resolution at the same pH as its highest measured activity (pH 5.5). In the active site, a water molecule, bridging between the catalytic Arg15 and the reaction-product analogue sulfate, might be representative of the water molecule attacking the phospho-cysteine intermediate in the second step of the reaction mechanism.

Keywords: Enterobacteriaceae; Erwinia amylovora; amylovoran; fire blight; hydrolases; kinetics; low-molecular-weight protein tyrosine phosphatase.

Figure 1

Alignment of LMW-PTPs. Sequence alignment of LMW-PTPs for which X-ray diffraction structures are available (the sequence names consist of the protein name followed by the source organism). The secondary-structural elements in AmsI are shown at the top. Conserved residues are highlighted in red. The characters in red represent the conservative residues. This figure was created using the ESPript server (Robert & Gouet, 2014 ▸).

Figure 2

AmsI structure. Cartoon representation of the E. amylovora low-molecular-weight protein tyrosine phosphatase AmsI. The secondary-structure elements are named and coloured according to their N- to C-­terminal position within the protein chain (e.g. from α1 to α5). This image was prepared with the PyMOL molecular-graphics system (v.1.7; Schrödinger).

Figure 3

AmsI active site. Residues 10–16 of the P-loop, the sulfate (SO₄) and the water molecule (H₂O; HOH2017) in the active site are shown in ball-and-stick representation. Atoms are coloured as follows: carbon, grey; oxygen, red; nitrogen, blue; sulfur, yellow; hydrogen, white. The water H atoms are included in the model. Hydrogen bonds are represented as dotted yellow lines. This image was prepared with the PyMOL molecular-graphics system (v.1.7; Schrödinger).