A multifunctional Pasteurella multocida sialyltransferase: A powerful tool for the synthesis of sialoside libraries

Article abstract of DOI:10.1021/ja0561690

A multifunctional sialyltransferase has been cloned from Pasteurella multocida strain P-1059 and expressed in E. coli as a truncated C-terminal His₆-tagged recombinant protein (tPm0188Ph). Biochemical studies indicate that the obtained protein

Full text of DOI:10.1021/ja0561690

Published on Web 11/25/2005
A Multifunctional Pasteurella multocida Sialyltransferase: A Powerful Tool for
the Synthesis of Sialoside Libraries
Hai Yu,† Harshal Chokhawala,† Rebekah Karpel,† Hui Yu,† Bingyuan Wu,‡ Jianbo Zhang,§
Yingxin Zhang,∇ Qiang Jia,# and Xi Chen*,†
Department of Chemistry, UniVersity of California, One Shields AVenue, DaVis, California 95616
Received September 7, 2005; E-mail: chen@chem.ucdavis.edu
Sialic acid (Sia)-containing structures play important roles in
cellular recognition and communication.1 Chemical sialylation is
considered as one of the most difficult glycosylation reactions due
to the hindered tertiary anomeric center and the lack of a
neighboring participating group in sialic acids.2 Enzymatic sialy-
lation catalyzed by sialyltransferases (SiaTs), especially those from
bacterial sources,2a,3 is an attractive alternative.4 A highly active
Figure 1. Multiple functions of a Pm sialyltransferase (tPm0188Ph).
sialyltransferase with broad substrate specificity will be a powerful
tool for broadening the application of chemoenzymatic approaches
in synthesizing diverse naturally occurring or structurally modified
sialosides. These compounds are invaluable probes for studying
the important biological roles of sialosides.5 Herein we report the
discovery and the characterization of a highly soluble and extremely
active multifunctional sialyltransferase from Pasteurella multocida
(Pm). Its application in a one-pot three-enzyme system for efficient
synthesis of diverse sialoside libraries is also described.
BLAST search using the amino acid sequence of a Photobac-
terium damsela R2,6SiaT (Pd2,6SiaT)6 as a probe identified a
putative SiaT (GenBank accession number AAK02272) encoded
by gene Pm0188 from Pasteurella multocida genomic strain Pm70.
The protein shared 37% identity and 57% amino acid similarity to
the sialyltransferase domain of the Pd2,6SiaT. A homologue of gene
Pm0188 was amplified from the chromosome DNA of Pm strain
P-1059 (ATCC 15742) and cloned into a pET23a(+) vector. DNA
sequencing indicated that the obtained gene had 10 base differences
compared to the published Pm0188 gene sequence, which led to
three amino acid differences in the deduced protein (D105N,
R135Q, G295E). The protein was named Pm0188Ph (Pm0188
Protein homologue).
Topology analysis identified a hydrophobic N-terminal domain
(1-25 aa) in the Pm0188Ph. Ni2+-column purified C-terminal His6-
tagged Pm0188Ph gave two protein bands in SDS-PAGE gels.
The smaller size band represented 95% of the total amount of the
purified protein. N-terminal amino acid sequencing of this band
indicated that the N-terminal 25 amino acid residues of the full
length protein had been cleaved off.
was (1) an R2,3SiaT that transferred a Sia residue from CMP-Sia
to galactosides to form R2,3-sialyl linkages efficiently in a wide
pH range (pH 6.0-10.0) with an optimal activity at pH 7.5-9.0;
(2) an R2,6SiaT that formed R2,6-linkages much less efficiently at
pH 4.5-7.0; (3) a sialidase that specifically cleaved R2,3-sialyl
linkages but left R2,6-sialyl linkages intact (optimal pH ) 5.0-
5.5); and (4) a trans-sialidase that transferred the Sia residue from
R2,3-linked sialyl galactosides (not R2,6-linked sialyl galactosides)
to another galactoside (optimal pH ) 5.5-6.5). A divalent metal
ion, such as Mg2+ or Mn2+, was not required for any of these four
activities.
The expression level (6000 U/L purified protein) and the specific
activity (60 U/mg protein) of the tPm0188Ph R2,3SiaT activity were
the highest among all SiaTs known to date [1 U ) 1 µmol of
product formed from CMP-N-acetylneuraminic acid (CMP-
Neu5Ac, donor) and 4-methylumbelliferyl-â-D-lactoside (LacMU,
acceptor) per minute at 37 °C, pH 8.5]. The apparent kinetic data
were obtained for CMP-Neu5Ac (KM ) 0.44 mM, kcat ) 32 s-1
)
and LacMU (KM ) 1.4 mM, kcat ) 47 s-1). The kcat/KM of LacMU
(33 s-1 mM-1) for the tPm0188Ph R2,3-SiaT was about 90-fold
higher than that (22 min-1 mM-1) of 8-aminopyrene-1,3,6-
trisulfonic acid-labeled lactose for N. meningitidis R2,3SiaT.3a
The efficiency and the flexibility of the tPm0188Ph in the
synthesis of R2,3-linked sialoside libraries were tested using a one-
pot three-enzyme system (Scheme 1). In this system, CMP-Sia
Scheme 1. A One-Pot Three-Enzyme System for the Synthesis of
Sialosides
A truncated protein (tPm0188Ph) lacking the N-terminal 2-25
amino acid residues of the Pm0188 Ph was sub-cloned as a C-His6-
tagged protein. The obtained tPm0188Ph was highly soluble and
could be expressed (37 °C for 3 h with shaking at 250 rpm) in
high yields in E. coli by isopropyl-1-thio-â-D-galactopyranoside
(0.1 mM) induction. About 100 mg of tPm0188Ph could be
routinely purified from the cell lysate obtained from a 1 L E. coli
culture. SDS-PAGE and gel filtration analyses indicated that the
tPm0188Ph existed as a monomer in solution.
The recombinant tPm0188Ph was a multifunctional enzyme for
which four types of functions (Figure 1) have been identified. It
derivatives were generated in situ from sialic acid precursors
catalyzed by a recombinant E. coli K12 sialic acid aldolase and a
recombinant N. meningitidis CMP-Sia synthetase.5a Under reaction
conditions used (37 °C, pH 8.5, 1-2 h), no significant sialidase or
R2,6SiaT activity was observed.
† University of California, Davis.
‡ Current address: Neose Technologies, Inc., Horsham, PA.
§ Current address: Department of Chemistry, East China Normal University,
China.
As shown in Table 1, the tPm0188Ph had the most flexible donor
substrate specificity among all bacterial SiaTs reported to date. It
∇ Current address: Incyte Corp., Experimental station, Wilmington, DE.
# Current address: Drug Source Company, Shanghai, China.
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J. AM. CHEM. SOC. 2005, 127, 17618-17619
10.1021/ja0561690 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Synthesis of Sialoside Libraries Using tPm0188Ph in a
One-Pot Three-Enzyme System, as Shown in Scheme 1
The tPm0188Ph R2,3SiaT also had relaxed acceptor specificity.
As shown in Table 1, N-acetyllactosamine (entry a), lactose (entries
b and l), and â-lactosides with methyl (entries f, q, t, and u), azido
(entries c and p), azidopropyl (entries d, m, o, and v), or
4-methylumbelliferyl (entries e, r, and s) aglycons were excellent
acceptors. Galactose (entry g) and â-galactosides (entries h, i, j,
and n) were good acceptors. Similar to that reported for the N.
meningitidis R2,3SiaT,3a an R-galactoside (e.g., R-methylgalacto-
side, entry k) was an acceptor for tPm0188Ph. N-Acetylgalac-
tosamine (GalNAc) and its derivatives, such as RGalNAcProN3 and
âGalNAcProN3, however, were not acceptors.
By controlling the pH value of reaction, tPm0188Ph could be
used in synthesizing R2,6-linked sialosides. For example, Neu5AcR2,-
6LacMU was obtained in 5.5% yield using a one-pot two-step
reaction in which CMP-Neu5Ac was synthesized at pH 8.8 in the
first step catalyzed by the sialic acid aldolase and the CMP-Sia
synthetase. After acidification of the reaction to pH 5.5, tPm0188Ph
was added and the trisaccharide was produced in the second step
without isolating the CMP-Neu5Ac intermediate.
The application of the trans-sialidase activity of tPm0188Ph in
the synthesis of sialosides was also tested. In this case, Neu5AcR2,-
3LacMU was obtained in 36% yield from Neu5AcR2,3-lactose and
LacMU.
In conclusion, due to its broad substrate specificity, high
solubility, high expression level, and multifunctionality, the newly
discovered P. multocida SiaT tPm0188Ph is an extremely powerful
tool for synthesizing structurally diverse sialosides to understand
their important biological functions. The unusual multifunctionality
of the enzyme as an R2,3SiaT, an R2,6SiaT, an R2,3-sialidase, and
an R2,3-trans-sialidase also provides solid evidence for the
complexity of bacterial sialylation.
Acknowledgment. This work was supported by Mizutani
Foundation for Glycoscience, New Faculty Research Grant, and
start-up funds from the Regents of the University of California.
Supporting Information Available: Experimental details for
cloning, expression, purification, and characterization of tPm0188Ph,
and details for chemoenzymatic synthesis of sialosides. This material
is available free of charge via the Internet at http://pubs.acs.org.
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