Journal of the American Chemical Society
COMMUNICATION
sialyl cation transition state less easily held by NanC, and it is
therefore released as the transition-state analogue Neu5Ac2en
(
Figure 3). Mutations of Phe396 alone or with Trp581 allow
NanC to produce some Neu5Ac, as well as Neu5Ac2en (Figures
S4.3, S4.4).
The subsequent Neu5Ac2en hydration reaction probably only
occurs at high concentrations of Neu5Ac2en, and presumably
proceeds via a similar mechanism to that previously reported for
13
catalysis by C. perfringens NanI. Briefly, the electrons in the
nucleophilic π bond could attack the proton of the catalytic aspartic
acid (Asp315), resulting in the formation of an oxocarbonium ion
intermediate. Without any lactose covering on top, water molecules
could be readily picked up and activated by Asp315. The water
attacks the positive charge at C-2 position to produce a protonated
intermediate, which transfers a proton back to Asp315, thus,
releasing Neu5Ac. An Asp315Ala mutant retains only 2% activity
and lacks Neu5Ac2en hydrating capacity (Figures S4.5, S4.6),
further confirming the role of Asp315 in catalysis. To discount
the possibility of an elimination reaction, a thiosialoside substrate
was examined with NanC by NMR, but this showed no activity
(
Figure S4.7).
The findings reported here, as summarized in Figure 3, add
further evidence to the currently proposed sialidase mechanisms.
By adopting distinct catalytic mechanisms, NanA, NanB, and
NanC might work together to coordinate the sialidase action
associated with pneumococcal virulence. Since the pneumococ-
cal sialidases are potential drug targets, our findings show that, for
each of the three distinct sialidases, different strategies may need
to be considered for ongoing structure-based drug design.
’
ASSOCIATED CONTENT
Supporting Information. Experimental details, H NMR
1
S
Figure 3. The proposed mechanisms of NanA, NanB, and NanC.
b
spectra and sequence alignment data. This material is available
ring that acts as an acid/base, as well as a hydrophobic pocket
accommodating the C-5 acetamido group (Figure 2b, Figure S3,
Table S1). These are the features required for catalysis, which
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
comprises a distortion of the sugar ring to a boat conformation
2
Corresponding Author
(
B ) upon binding and subsequent formation of a transition state
5
4
with the formation of the H half-chair oxocarbonium ion or
covalently bonded intermediate in a C chair form (Figure 3).
5
2
5
Present Addresses
Department of Respiratory Medicine, The Second Affiliated
Given that the three pneumococcal sialidases have the requisite
conserved catalytic residues to cleave sialosides, it is reasonable to
conclude that it is the final product formation step which is different
for each of the sialidases. As depicted in Figure 3, depending on
attack at C-2 position of the oxocarbonium ion by different
nucleophiles, such as a water molecule in hydrolytic sialidase, or
the O-7 atom of the sialyl cation in IT trans-sialidase or another
carbohydrate acceptor such as βGal in trans-sialidase, the final
products could be Neu5Ac, Neu2,7-anhydro5Ac, or Neu5Ac-βGal.
Here, S. pneumoniae NanA and NanB follow the mechanisms of a
hydrolytic sialidase and an IT trans-sialidase, respectively.
#
Hospital, Nanchang University, 1 Minde Road, Nanchang, China
3
30006.
’ ACKNOWLEDGMENT
G.X. was supported by BioCryst Pharmaceuticals, Inc.
Birmingham, AL). P.W.A. and M.R.O. were funded by the EU
Pneumopep). Authors thank Dr. Uli Schwarz-Linek, Dr. Robert
(
(
Hagan, and Prof. Jim Naismith for kind help and useful discus-
sions.
In the first reaction to produce Neu5Ac2en, NanC might also
adopt the generalized mechanism involving the formation of a
transition-state sialyl cation or a covalently sialylated enzyme
intermediate (Figure 3). However, in contrast to NanA and NanB,
there must be no effective nucleophilic attack happening in the
active site of NanC. This may in part be attributable to the
hydrophobic environment created by residues stacking around
the catalytic cleft, as well as the leaving lactose group covering on
top. Furthermore, NanC can only form limited hydrogen bonds
’
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with Neu5Ac2en with a low K of ∼3 mM. This also makes the
i
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dx.doi.org/10.1021/ja110733q |J. Am. Chem. Soc. 2011, 133, 1718–1721