P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation

The Molecular Mechanism of Alginate O-Acetylation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic, gram-negative pathogen that is involved in a variety of infections. The bacterium secretes and chemically modifies alginate to form a protective biofilm matrix. One protein implicated in the O-acetylation of alginate is AlgJ. In this study, we have successfully crystallized AlgJ and determined its structure to 1.83Å. The structure reveals that AlgJ is a SGNH hydrolase-like protein. AlgJ possesses a Ser-Asp-His catalytic triad which suggests that AlgJ is catalytically active. The structure of AlgJ demonstrated that the previously identified conserved residues of the AlgJ signature motifs form a local network with the proposed catalytic triad and are important for alginate O-acetylation in vivo. We have assayed the proposed Ser-Asp-His catalytic triad in vitro and observed impairment in acetyl esterase activity. In vitro binding studies demonstrated that AlgJ was unable to bind polymannuronate and that was it not capable of O-acetylating the polymer.MAS

Apparent association constants (<i>K</i>_a) for <i>Pa</i>AlgX_27–474 and <i>Pa</i>AlgJ_79–379 for short polymannuronic oligosaccharides at 298 K and pH 7 determined by the direct ESI-MS assay.

<p>NB: No Binding.</p><p>*Ligand name as referenced in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004334#ppat.1004334-Walvoort2" target="_blank">[78]</a>.</p><p>Apparent association constants (<i>K</i>_a) for <i>Pa</i>AlgX_27–474 and <i>Pa</i>AlgJ_79–379 for short polymannuronic oligosaccharides at 298 K and pH 7 determined by the direct ESI-MS assay.</p

ESI mass spectra to examine polymannuronic acid binding.

<p>ESI mass spectra acquired for aqueous ammonium acetate (50 mM) solutions of (A) <i>Pa</i>AlgX_27–474 (P) (4 µM) and 50 mM ManA₆); (B) <i>Pa</i>AlgX_27–474(P) (4 µM) and 50 mM ManA₁₂); (C) <i>Pa</i>AlgJ_79–379 (P) (10 µM) and 50 mM ManA₆); (D) <i>Pa</i>AlgJ_79–379 (P) (10 µM) and 50 mM ManA₁₂). A reference protein scFv (4 µM) was added into the solutions of AlgX, Lyz was added as P_ref to AlgJ solutions. Ion peaks corresponding to higher charge states of AlgJ are labelled as filled red circles. P_ref refers to the reference protein scFv.</p

Structure and topology of <i>Pp</i>AlgJ_75–370.

<p>(A) Cartoon representation of <i>Pp</i>AlgJ_75–370 with secondary structural elements labelled (α: α-helix; β: β-strand; and t: 3₁₀ helix). Residues at discontinuous points in the structure due to poor observed electron density are labelled and coloured red. The N- and C-termini of the protein are labelled N and C, respectively, and the terminal residue is coloured red. (B) Topology model of the <i>Pp</i>AlgJ_75–370 structure with secondary structural elements and termini labelled as in panel (A).</p

Proposed model for the <i>O</i>-acetylation of alginate.

<p>AlgI receives the currently unidentified acetyl donor molecule on its cytoplasmic face and shuttles the acetate or acetyl donor molecule through the inner membrane, where it is transferred to either AlgJ and/or AlgF. The acetyl donor may or may not be transferred between AlgJ and AlgF. From its intermediate location, the acetyl donor molecule is transferred to AlgX, which subsequently <i>O</i>-acetylates the alginate polymer. Although absolutely required for alginate <i>O</i>-acetylation the function of AlgF is currently unknown. We have demonstrated that AlgJ has activity on acetylated substrates thereby suggesting it fulfills a more direct role in the O-acetylation process.</p

Architecture of the proposed <i>Pp</i>AlgJ_75–370 active site compared to AlgX.

<p>(A) Superposition of the cartoon representations of <i>Pp</i>AlgJ_75–370 (blue) and <i>Pa</i>AlgX_44–344 (grey). The conserved residues implicated in catalysis in SGNH hydrolases are displayed as sticks and coloured either red (<i>Pp</i>AlgJ) or grey (AlgX). The dashed box indicates the region where the conserved residues are located. (B) Enlarged figure highlighting the similarity of the residues in the active site region in both proteins. Red stick residues and text identifies the conserved residues from <i>Pp</i>AlgJ whereas the grey sticks and black text identifies residues from AlgX. Yellow dashed lines represent hydrogen bonding between members of the catalytic triad with values in angstroms (Å). (C) Sequence alignment of various members of the SGNH hydrolase superfamily depicting four consensus blocks shared by members. <i>Pp</i>AlgJ: <i>P. aeruginosa</i> O-acetyltransferase (Q88ND3), PaAlgX: <i>P. aeruginosa O</i>-acetyltransferase (Q51372) EstA: <i>Lactobacillus helveticus</i> arylesterase (Q9LAH7), EtpA: <i>Vibrio mimicus</i> arylesterase (Q07792). Conserved residues are masked in blue (absolutely conserved) or green (conserved in most members). Residues masked in purple correspond to the conserved tyrosine in AlgJ and AlgX that are proposed to be analogous in function to asparagine in other superfamily members. Brackets correspond to Uniprot identifiers.</p

Specific activities of wild-type and catalytic triad mutants of <i>Pp</i>AlgJ_75–370 and <i>Pa</i>AlgJ_79–379.

<p>Specific activities were measured at an initial concentration of 3.0 mM ACC. WT and specific mutants for each AlgJ construct are labelled below the x-axis. Specific activity upon the y-axis was measured as µmol min⁻¹ mg⁻¹.</p

The AlgJ signature motifs.

<p>(A) Cartoon representation of <i>Pp</i>AlgJ_75–370 with residues from the signature motif represented as sticks. The AlgJ signature motif residues have been coloured according to the amount of alginate acetylation observed <i>in vivo</i> in each alanine variant relative to WT AlgJ. Yellow represents impairment of between 50–60% acetylation; orange represents strong impairment with only 6–25% acetylation observed; and red represents ablation or between 0–5% observed acetylation. Residues represented as grey sticks are proposed to be involved in interactions with the AlgJ signature motif residues but have not been characterized <i>in vivo</i>. (B) Selected residues from the box area in panel A that participate in or are proposed to be involved in the hydrogen bonding network and are coloured as described in (A). Hydrogen-bonds are depicted as green dashes with distance given in angstroms (Å). (C) Residues participating in hydrophobic and van der Waals interactions with W193 (shown in orange) are depicted in grey. Hydrophobic and van der Waals interactions less than 3.7 Å are depicted with blue dashes.</p

AlgX_27–474 is an <i>O</i>-acetyltransferase.

<p>Mass spectra of the acetyltransferase reactions in the presence (red, line a) and absence (blue, line b) of <i>Pa</i>AlgX_27–474. Major peaks are labelled: ManA10, mannuronic acid decamer; ManA10+Na, mannuronic acid decamer plus sodium; ManA10+OAc, mannuronic acid decamer plus acetate; ManA10+Na+OAc, mannuronic acid decamer plus sodium and acetate; ManA10+2 OAc, mannuronic acid decamer plus two acetates. The observed molecular weights of each product are denoted under their respective labels. The addition of acetate adds 42.01 Da to the mass of the polymer. The data has been deconvoluted and centroided using Agilent MassHunter software and represents uncharged exact masses.</p