Structural studies on the sialidases from Streptococcus pneumoniae and Pseudomonas aeruginosa

The sialidases are a group of glycosyl hydrolases that specifically remove terminal\nsialic acid (Neu5Ac) residues from various glycans. In the two common human\npathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these\nenzymes have been shown to be key virulence factors directly involved in bacterial\ncolonization and infection. However, little is known about their detailed structural and\nmechanistic features and lack of this information significantly slows down the progress\nof new drug discovery targeting these enzymes. Therefore, we embarked structural and\nkinetic studies towards the three distinct sialidases (designated as NanA, NanB and\nNanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA)\nfrom P. aeruginosa.\nFull-length NanA failed to crystallize due to the presence of some natively disordered\nregions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was\ncrystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with\nclose contacts between the two monomers. The second pneumococcal sialidase NanB\nonly shares 24% sequence identity with NanA. Crystal structure of NanB was also\ndetermined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In\ngeneral, the core catalytic domain of both CNanA and NanB adopts the classic six-\nbladed β-propeller fold (or called sialidase fold), with a set of highly conserved\nresidues stacking around the proposed active sites. NanC is a close homologue of\nNanB, sharing over 50% sequence identity. However, NanC crystallization is not\nsuccessful so far. To compare the three sialidases in more detail, a computational\nNanC model was made based on the structure of NanB. Mapping of the active sites of\nCNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the\nprobe. Although sharing many common features, NanA, NanB and NanC present\ndifferent topologies around the catalytic centre, give these enzymes a high level of\ndiversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR\nstudies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to\nbe an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles\nmultiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3-\nsialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S.\npneumoniae thus expresses NanA, NanB and NanC for disparate but cooperative roles. Such a working pattern of three sialidases in one microbe is unusual in nature, which\nmight be essential for pneumococcal pathogenesis at various stages. Based on the\ncrystal structures of CNanA and NanB, preliminary work towards S. pneumoniae\nsialidases inhibitor design is under way, in which, a variety of techniques, such as the\nfluorescence-based thermal shift assay, NMR spectroscopy, computational docking\nand X-ray crystallography, are incorporated in.\nThe crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a\nunique trimer in crystal that is associated, in part, by the immunoglobulin-like\ntrimerization domain around a three-fold crystallographic axis. The core catalytic\ndomain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase\nactivity was detected with this enzyme. In addition, two key catalytic residues\nincluding one of the arginine in the arginine triad and the acid/base catalyst aspartic\nacid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate\nselectivity towards pseudaminic acid, which is a specific carbohydrate superficially\nsimilar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed\nmutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA\ninto a poor sialidases. Moreover, the crystal structure of PaNA also indicates that\nHis45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic\nacid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315\nto be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA\ncould be a pseudaminidase with structural and mechanistic variations. This enzyme,\ntogether some other uncharacterized fellow proteins, might form a novel subclass in\nthe sialidase superfamily.\nThe various findings in the current projects provide meaningful insights towards\nseveral sialidases that have been linked to bacterial virulence, which may contribute to\na more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.