Structural studies on the sialidases from Streptococcus pneumoniae and Pseudomonas aeruginosa
Abstract
The sialidases are a group of glycosyl hydrolases that specifically remove terminal
sialic acid (Neu5Ac) residues from various glycans. In the two common human
pathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these
enzymes have been shown to be key virulence factors directly involved in bacterial
colonization and infection. However, little is known about their detailed structural and
mechanistic features and lack of this information significantly slows down the progress
of new drug discovery targeting these enzymes. Therefore, we embarked structural and
kinetic studies towards the three distinct sialidases (designated as NanA, NanB and
NanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA)
from P. aeruginosa.
Full-length NanA failed to crystallize due to the presence of some natively disordered
regions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was
crystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with
close contacts between the two monomers. The second pneumococcal sialidase NanB
only shares 24% sequence identity with NanA. Crystal structure of NanB was also
determined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In
general, the core catalytic domain of both CNanA and NanB adopts the classic six-
bladed β-propeller fold (or called sialidase fold), with a set of highly conserved
residues stacking around the proposed active sites. NanC is a close homologue of
NanB, sharing over 50% sequence identity. However, NanC crystallization is not
successful so far. To compare the three sialidases in more detail, a computational
NanC model was made based on the structure of NanB. Mapping of the active sites of
CNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the
probe. Although sharing many common features, NanA, NanB and NanC present
different topologies around the catalytic centre, give these enzymes a high level of
diversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR
studies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to
be an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles
multiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3-
sialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S.
pneumoniae 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
might be essential for pneumococcal pathogenesis at various stages. Based on the
crystal structures of CNanA and NanB, preliminary work towards S. pneumoniae
sialidases inhibitor design is under way, in which, a variety of techniques, such as the
fluorescence-based thermal shift assay, NMR spectroscopy, computational docking
and X-ray crystallography, are incorporated in.
The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a
unique trimer in crystal that is associated, in part, by the immunoglobulin-like
trimerization domain around a three-fold crystallographic axis. The core catalytic
domain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase
activity was detected with this enzyme. In addition, two key catalytic residues
including one of the arginine in the arginine triad and the acid/base catalyst aspartic
acid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate
selectivity towards pseudaminic acid, which is a specific carbohydrate superficially
similar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed
mutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA
into a poor sialidases. Moreover, the crystal structure of PaNA also indicates that
His45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic
acid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315
to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA
could be a pseudaminidase with structural and mechanistic variations. This enzyme,
together some other uncharacterized fellow proteins, might form a novel subclass in
the sialidase superfamily.
The various findings in the current projects provide meaningful insights towards
several sialidases that have been linked to bacterial virulence, which may contribute to
a more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.
Type
Thesis, PhD Doctor of Philosophy
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