Structural Studies on the Pseudomonas aeruginosa Sialidase-Like Enzyme PA2794 Suggest Substrate and Mechanistic Variations Guogang Xu 1 , Charlotte Ryan 1 , Milton J. Kiefel 2 , Jennifer C. Wilson 2 and Garry L. Taylor 1 ⁎ 1 Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife KY16 9ST, UK 2 Institute for Glycomics, Griffith University, PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia Received 10 November 2008; received in revised form 23 December 2008; accepted 31 December 2008 Available online 10 January 2009 Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical β-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/ base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site. Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved. Edited by G. Schulz Keywords: sialidase; neuraminidase; pseudaminic acid; drug design Introduction There is increasing evidence that bacteria use a variety of carbohydrates to decorate both their lipo- polysaccharides (LPS) and certain of their proteins. 1–3 One unusual carbohydrate is the nine-carbon sugar 5,7-diamino-3,5,7,9-tetradeoxy L-glycero-L-manno- nonulosonic acid (pseudaminic acid, or Pse5Ac7Ac, 2 in Fig. 1). Pseudaminic acid is structurally related to sialic acid, Neu5Ac (1 in Fig. 1), but has a different stereochemistry at the 5-, 7-, and 8-positions and was first discovered in the LPS of Pseudomonas aeru- ginosa and Shigella boydii. 4,5 The flagella of Campy- *Corresponding author. E-mail address: glt2@st-andrews.ac.uk. Abbreviations used: LPS, lipopolysaccharides; PaNA, Pseudomonas aeruginosa sialidase/neuraminidase; SIRAS, single isomorphous replacement with anomalous scattering; Ig, immunoglobulin; TNF, tumor necrosis factor; PDB, Protein Data Bank; MUAN, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid; 4-MU, 4-methylumbelliferone; 3SL, α2,3-sialyllactose; PEG, polyethylene glycol. doi:10.1016/j.jmb.2008.12.084 J. Mol. Biol. (2009) 386, 828–840 Available online at www.sciencedirect.com 0022-2836/$ - see front matter. Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved.