The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines a third class of sulfatases

Abstract

Pseudomonas aeruginosa is both a ubiquitous environmental bacterium and an opportunistic human pathogen. A remarkable metabolic versatility allows it to occupy a multitude of ecological niches, including wastewater treatment plants and such hostile environments as the human respiratory tract. P. aeruginosa is able to degrade and metabolize biocidic SDS, the detergent of most commercial personal hygiene products. We identify SdsA1 of P. aeruginosa as a secreted SDS hydrolase that allows the bacterium to use primary sulfates such as SDS as a sole carbon or sulfur source. Homologues of SdsA1 are found in many pathogenic and some nonpathogenic bacteria. The crystal structure of SdsA1 reveals three distinct domains. The N-terminal catalytic domain with a binuclear Zn2+ cluster is a distinct member of the metallo-beta-lactamase fold family, the central dimerization domain ensures resistance to high concentrations of SDS, whereas the C-terminal domain provides a hydrophobic groove, presumably to recruit long aliphatic substrates. Crystal structures of apo-SdsA1 and complexes with substrate analog and products indicate an enzymatic mechanism involving a water molecule indirectly activated by the Zn2+ cluster. The enzyme SdsA1 thus represents a previously undescribed class of sulfatases that allows P. aeruginosa to survive and thrive under otherwise bacteriocidal conditions.

Fig. 1.

Structure of SdsA1. (A) A cartoon representation of the SdsA1 dimer. Structural domains are color-coded: N-terminal domain, blue; dimerization domain, green; C-terminal domain, orange-red. A disordered loop connecting α-helices α15 and α16 is indicated by a dotted line, and Zn ions are represented by yellow spheres. (B) The C-terminal domain of SdsA1. Amino acid side chains lining the hydrophobic groove are shown in stick representation. (C) A large water-filled cavity within the dimerization domain of SdsA1. Van der Waals surfaces of enclosed water molecules (red spheres) are shown in translucent gray. (D) Schematic representation of the SdsA1 secondary structure: α-helices are represented by circles, β-strands by triangles, loops by lines, and Zn ions by yellow spheres. The second gray-colored dimerization domain is included to emphasize the extensive intertwining of the SdsA1 dimer.

Fig. 2.

The catalytic domain of SdsA1. (A) Cartoon with coloring as in Fig. 1. Side chains coordinating Zn ions (yellow spheres) are shown as sticks. The active-site dome (gray) is not conserved in other MBL fold enzymes. The sulfate recognition loop βN/α8 is shown in purple. (B) Structure-based sequence alignment of SdsA1 and B. fragilis β-lactamase (PDB: 1A7T). Secondary structure elements of SdsA1 are depicted by rods and arrows. Conserved residues are marked in red; those conserved in all enzymes (see Table 2, which is published as supporting information on the PNAS web site) are marked by asterisks, and metal ion ligands are marked by yellow circles.

Fig. 3.

Active site of SdsA1. (A) The Zn²⁺-binding site. Zn ions are depicted as yellow spheres, water molecules or hydroxyl ions as red spheres, and metal-binding residues as stick models. Selected hydrogen bonds and interactions are indicated by gray dotted lines, and distances are provided in angstroms. Backbone colors indicate temperature factors: red, high structural flexibility; blue, low structural flexibility. Loop βL/βM adopts two distinct conformations depending on Zn2 occupancy. Closed conformation, red (high B factor); open conformation, green. Corresponding conformations of Tyr-325 are also shown. The van der Waals surface of Ile-239 is shown in translucent orange. Zn²⁺ ligands of SdsA1 and MBL proteins listed in Table 2 are compared (Inset). (B) Active site of SdsA1 with SDS analogue 1DA (vivid colors) superimposed on reaction product structures, SO₄²⁻ (light blue) and 1DO (light green). Zn²⁺ ligands are shown as thin gray sticks, and substrate/product-binding residues are shown as thicker, colored sticks. A dotted yellow circle indicates the equatorial plane of the distorted trigonal-bipyramidal configuration of S_1DA. (B Inset) 2F_o − F_c electron density of 1DA (1σ, blue) depicts the relative location of the proposed nucleophile W₂ relative to S_1DA. (C) A schematic representation of the 1DA bound to the active site. H bonds and salt bridges are marked by dotted lines, hydrophobic interactions are marked by gray arcs, and distances are in angstroms.

Fig. 4.

The hydrophobic chute connecting the C-terminal domain with the active site of SdsA1. The van der Waals surface of SdsA1 (colored) has been cut away to reveal the hydrophobic chute; the molecular interior is colored gray. Zn ions (yellow spheres) mark the active site. Selected amino acids are marked. (A) Predominantly hydrophobic residues (gray) line the chute, whereas polar (light blue), basic (blue), and acidic (red) amino acids mark the active site or the molecular surface. (B) Amino acids lining the chute are strongly conserved (green) in contrast to the molecular surface (gray). Bound 1DA is shown in ball-and-stick representation.