Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7

Abstract

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.

Figure 1

Esterase activity of NanS. (a) A range of substrates that were used to define the substrate selectivity profile (see also Table I); (b) Mutant activity compared to wild type NanS (see Table II). The bars on the graph indicate standard deviation. The substrates were butyl acetate, phenyl acetate and pNP-acetate. H301N and S19A show no activity while the remaining four mutants show varying levels of activity.

Figure 2

A topological representation of NanS. α helices are shown as cylinders, β sheets as arrows, which show their direction and random coil as lines. The short section (216–218) that was not observed in the crystal structure is shown as a dashed line. Image created using PDBSum (http://www.ebi.ac.uk/pdbsum/)

Figure 3

Structure of NanS and comparison with other SGNH enzymes. (a) Stereo view showing cartoon representation of NanS colored by secondary structure elements, Red represents α helices, yellow β sheet, and green random coil. The active site residues, His301 and Ser19, are shown in stick mode to indicate the location of the substrate binding site. Semitransparent molecular surface is colored wheat; (b) superposition of NanS (family II), shown in blue, and serine esterase SsEst (family I, PDB code 1ESC), shown in yellow. They are in the same orientation as the monomer above; (c) hydrogen bond network (green dashed lines) in the active site pocket in NanS. (d) All residues that are involved in the hydrogen bonding network of the oxyanion hole in NanS are shown. Magenta dashed line shows the interaction between the catalytic residues. This network is conserved in all family II members. For both (c) and (d), residues are shown as sticks and colored according to convention. Water molecules are shown as red spheres. All images were created using Pymol (www.pymol.org) [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com].

Figure 4

Structure-based sequence alignment of several enzymes from Family I and Family II SGNH hydrolases. Representing Family II, which are shown above the line, are NanS, acetyl xylan esterase (AXE) from Clostridium acetobutylicum (PDB ID 2APJ) and the At4g34215 gene product (At4g) from Arabidopsis thaliana (PDB ID 1ZMB). The sequences that represent Family I, below the line, are serine esterase (SsEst) from Streptomyces scabies (PDB ID 1ESC), rhamnogalacturonan acetylesterase (RGAE) from Aspergillus aculeatus, (PDB ID 1DEO) and thioesterase I (TAP) from E. coli (PDB ID 1JRL). Each conserved Block is labeled. White letter in a black box indicate residues that are conserved in all sequences. Bold boxed letters indicate conserved residue types in some of the sequences.