Download The Terminal Enzymes of Sialic Acid Metabolism: Acylneuraminate

Bioscience Reports, Vol. 19, No. 5, 1999
MINI
REVIEW
The Terminal Enzymes of Sialic Acid Metabolism:
Acylneuraminate Pyruvate-Lyases
Roland Schauer,1,2 Ulf Sommer,1 Dorothea Kruger,1 Henrieke van Unen,1
and Christina Traving1
The acylneuraminate pyruvate-lyase gene from Clostridium perfringens was sequenced and
found to be most similar to the lyase gene from Haemophilus influenzae. Both the recombinant clostridial enzyme and the native enzyme from pig kidney were purified in larger
amounts and characterized. The properties of the porcine lyase are similar to the microbial
ones. However, the much higher degree of similarity in comparison to the microbial
enzymes that was found between porcine lyase peptides and two putative mammalian lyase
sequences show that the latter form an own group apart from the microbial lyases. Actual
models of the acylneuraminate pyruvate-lyase reaction are discussed.
KEY WORDS: Acylneuraminate pyruvate-lyase; enzyme isolation; genetic relationship;
primary structure; reaction mechanism; sialic acid.
ABBREVIATIONS: DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EST,
expressed sequence tag; cDNA sequences obtained after reverse transcription of mRNA;
HIC, hydrophobic interaction chromatography; HPLC, high performance liquid
chromatography; N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid;
Neu4,5Ac2, N-acetyl-4-O-acetylneuraminic acid; Neu5,9Ac2, N-acetyl-9-Oacetylneuraminic acid; Neu2en5Ac, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid;
NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; PMSF,
phenyl methyl sulphonyl fluoride; SDS, sodium dodecyl sulfate.
INTRODUCTION
Acylneuraminate pyruvate-lyases [sialate(-pyruvate) lyases, sialic acid aldolases; EC
4.1.3.3] catalyze the cleavage of sialic acids into pyruvate and acylmannosamines.
Thus, they present the terminal enzymes of sialic acid catabolism (Schauer, 1982;
Schauer and Kamerling, 1997; Traving and Schauer, 1996). These lyases occur in
higher animals of the deuterostomate lineage, which also possess the corresponding
sialic acid substrates. In the cytosol, they take part in the turnover of sialic acids
and thereby prevent the recycling of these sugar molecules, which would otherwise
return to the cell surface after the transfer to glycoconjugates in the Golgi compartment. Furthermore, they have been found in microbial species (Muller, 1974; Nees
1
Institute of Biochemistry, Christian-Albrechts-Universitat, Olshausenstr. 40, D-24098 Kiel, Germany;
e-mail: [email protected]
2
To whom correspondence should be addressed.
373
0144-8463/99/1000-0373$16.00/0 © 1999 Plenum Publishing Corporation
374
Schauer, Sommer, Kruger, van Unen, and Traving
and Schauer, 1974), which also express sialic acids and the enzymes of sialic acid
synthesis and polymerization, e.g., several Escherichia coli strains (Vimr and Troy,
1985a, b). In these organisms, lyases are important for the regulation of the intracellular sialic acid concentration. Vimr and Troy (1985a) showed that in special
mutants lacking acylneuraminate pyruvate-lyase activity, the intracellular sialic acid
concentration reaches toxic levels. Finally, there are also some microbial species with
the lyase, which do not possess sialic acids, e.g., C. perfringens. Interestingly, these
species often live in close contact to higher animals, being their parasites or commensals. By expressing lyase activity, they get access to an additional carbon and energy
source (Nees and Schauer, 1974). As a prerequisite, the sialic acids have to be liberated from the host tissue by the action of sialidases.
There also exist reports about a synthetic function of lyases in vivo in certain
E. coli strains (Rodriguez-Aparicio et al., 1995, Ferrero et al., 1996). The enzymatic
synthesis of sialic acids and derivatives thereof by means of the lyase has been extensively worked out and the yield has been optimized (Wong et al., 1995). Therefore,
these enzymes might be involved in the development of inhibitors which act against
the sialidase of pathogens (von Itzstein et al., 1993). Furthermore, lyases are used
for the analysis of sialic acids, in order to confirm the identity of HPLC peaks in
the laboratory (Reuter and Schauer, 1994).
The best-known microbial lyase is from E. coli. It was purified and characterized
(Uchida et al., 1984; Aisaka et al., 1991), the protein crystallized (Aisaka et al.,
1991) and the X-ray structure resolved (Izard et al., 1994). It has also been overexpressed (Aisaka et al., 1987) and the primary structure is known (Ohta et al., 1985;
Kawakami et al., 1986). The primary structures are also known from the lyases of
H. influenzae (Fleischmann et al., 1995), Trichomonas vaginalis (Meysick et al., 1996)
and the partial sequence from Clostridium tertium (Grobe et al., 1998). There also
exist cDNA-sequences from human fetal heart (Hillier et al., 1995) and mouse
(Marra et al., 1996) in the EMBL-database that probably represent partial lyase
genes.
Here we report the purification, sequencing and expression of the lyase from C.
perfringens as well as the purification, characterization and first insight into the primary structure of the lyase from porcine kidney.
MATERIALS AND METHODS
To determine acylneuraminate lyase activity, the increasing amounts of Nacylhexosamines (Morgan-Elson-Assay; modified from Reissig et al., 1955) or pyruvate (Kawakami et al., 1986) or the decreasing amounts of sialic acids (Hara et al.,
1989) during the reaction were measured.
Materials and methods used for the purification, cloning and sequencing of the
clostridial lyase are described elsewhere (Traving et al., 1997). The clostridial lyase
gene was amplified and expressed as a fusion protein with a His-tag (Studier and
Moffat, 1986; Studier et al., 1990). A selected clone was grown on a larger scale and
the cells were harvested by centrifugation, resuspended in buffer and disrupted by
freezing and thawing. From the supernatant after centrifugation, the fusion was
isolated by metal chelate affinity chromatography (Hochuli, 1988) and characterized.
Acylneuraminate Pyruvate-Lyases
375
Lysine 161 of the clostridial lyase was exchanged against arginine, glutamine or
alanine, respectively, by site-directed mutagenesis (Barettino et al., 1993; Higuchi et
al., 1988; Ho et al., 1989). For characterization, the resulting clones were grown and
the mutant lyase proteins isolated as described above.
The lyase from pig kidney was isolated by homogenization of the tissue, heat
precipitation of the supernatant after centrifugation in the presence of sodium pyruvate, anion exchange chromatography (Q-Sepharose) and Hydrophobic Interaction
Chromatography (HIC) on an ethylagarose column (Sommer et al., manuscript in
prep.). The final purification was achieved by a second HIC run on an ET20 column
(perfusion chromatography), resulting in a specific activity of 1.4 U/mg of the pure
enzyme with a yield of 0.2%. After concentration, the protein solution was subjected
to SDS-PAGE and the protein band cut out. Peptide fragments of the enzyme were
separated and sequenced. Alternatively, the ethylagarose step was followed by native
PAGE. After cutting the gel into slices, the enzyme was eluted in potassium phosphate buffer containing dithiothreitol (DTT). This procedure gave a higher yield
(2%) of the pure enzyme with a specific activity of 1.5 U/mg. About 3mg of the
porcine lyase could be isolated.
The properties of the enzyme protein were determined in the usual manner
using the ET20-purified enzyme. The involvement of certain amino acids in the catalytic process was investigated by measuring the influence of amino acid-modifying
reagents on enzyme activity.
For the analysis of microbial primary structures see Traving et al., 1997; and
Grobe et al., 1998. EST sequences from mouse (WW162738; Marra et al., 1996) and
human fetal heart (A709930; Hillier et al., 1995) were derived from the EMBL database and translated in protein sequences. The alignment was checked by the program
husar 8.1 (Heidelberg).
RESULTS AND DISCUSSION
Data Deduced from the Investigation of Purified Proteins
Acylneuraminate pyruvate-lyases represent a rather uniform group of enzymes.
As can be seen from Table 1, in most of the studies the molecular mass of the
denatured enzyme was found to be about 33 kDa. From the much higher values,
which were obtained for the native molecular mass by gel filtration, it was concluded
that the enzyme, e.g., from E. coli, consists of three subunits. Similar values for the
native molecular mass were found for the lyases from pig kidney and C. perfringens.
Former studies with the electron microscope proposed the lyase of this species to be
a dimer of two 50 kDa subunits (Nees et al., 1976). The first crystal structure was
resolved of the E. coli lyase (Izard et al., 1994). It showed the enzyme to be a
tetramer. Such a structure was found also for the lyase from porcine kidney after
cross-linking experiments with dimethyl pimelimidate (Sommer et al., 1997). The
erroneous results from gel filtration were ascribed to an anomalous migration
behavior of the lyase proteins due to their special tetraedric shape (Izard et al.,
1994).
Table 2 shows a list of further lyase properties. While the pH-optimum in the
neutral range is no surprise and fits well with the probably cytoplasmic location of
376
Schauer, Sommer, Kruger, van Unen, and Traving
Table 1. Size and Subunit Structure of Acylneuraminate Pyruvate-Lyases
Clostridium
perfringens
Pig kidney
321,2
333,4
355,8
5010
3316
5811
3715
Deduced from
Gene structure
[kDa]
32.66
32.316
Native protein
[kDa]
1353
984
907,8
1058
9214
99.210
25013
Escherichia coli
Molecular mass
Denatured protein
[kDa]
Number of subunits
34,7,8
9
4
210$,
14
Trichomonas
vaginalis
Haemophilus
influenzae
312
3512
32.62
135–14515*
107–11015*
111
415
* Determined by cross-linking with dimethylpimelimidate; # determined by gel filtration; $ determined
by electron microscopy.
l2
1
7
Ohta et al., 1986.
Meysick et al., 1996.
Lilley et al., 1992.
13
2
8
Aisaka et al., 1991.
Barnett et al., 1971.
Lilley et al., 1998.
14
3
9
Izard et al., 1994.
DeVries and Binkley , 1972.
Ferrero et al., 1996.
15
4
10
Nees et al., 1976.
Sommer et al., in prep.
Uchida et al., 1984.
l6
5
11
Schauer and Wember, 1996.
Traving et al., 1997.
Kawakami et al., 1986.
6
Ohta et al., 1985.
the enzymes, their thermostability is a special feature that has even been used for
enrichment by heat precipitation of the majority of contaminating proteins. G.
Taylor (University of Bath), with whom we had a fruitful discussion about this
topic, stressed that a thermostable character has often been found for tetrameric
enzymes. He proposed salt bridges to be at least in part responsible for this property.
The optimum temperature is between 65°C (C. perfringens, recombinant enzyme)
and 80°C (E. coli). It is therefore tempting to speculate that the mechanism of lyase
reaction, discussed below, is very old as proposed by D. Grimmecke (Research Institute Borstel, Germany). Perhaps, the catalytic mechanism descended from some
ancestral reaction that was already invented by thermophilic organisms.
The kinetic data also do not diverge much between the enzymes studied so far.
The KM values are in the millimolar range. Most kinetic measurements were performed at 37°C. Only for the porcine lyase, KM and Vmax were also determined at
the optimum temperature (Sommer et al., 1997). The lower Michaelis constant of
this enzyme at 75°C may not be interpreted as a result of higher substrate affinity,
since the temperature dependence of each kinetic parameter influencing the idealized
Michaelis–Menten kinetics has to be considered.
The best substrate for the lyase reaction is Neu5Ac, but also Neu5Gc is cleaved
at half the rate (Table 3). Generally, substitution of the amino group of the substrate
has no great influence on the cleavage rate. Sialic acids with O-acetyl groups in the
side chain are also split to a lower degree and even Neu4,5Ac2 with some enzyme
preparations has been found to be an, although weak, substrate. The latter fact is
377
Acylneuraminate Pyruvate-Lyases
Table 2. Enzymatic Properties of Acylneuraminate Pyruvate-Lyases
Clostridium
perfringens
Escherichia coli
Pig kidney
Isoelectric point
4.71
4.56
5.511
pHopt.
7.21,8,9
7.53
7.77
6.5–7.06
7.24,10
7.6–8.011
topt.[°C]
Thermostability
Specific activity
[U/mg]
K M Neu5Ac [mmol]
V max
65–7011
70–8011
806
757
+2
+ 6,7
1
7
10
2.81
1.95
3.98
1.759
56.8
2.53
3.36
3.67
71.4 U/mg6
154. 5 U/mg 7
4,11
+
4
0.42
1.511
3.74
1.510
1.711#/111§
37.1mU 4
2.3 U/mg 11#
7.1 U/mg11§
# Determined at 37°C; § determined at 75°C
1
4
Nees et al., 1976.
Schauer and Wember, 8Comb and Roseman,
2
Kolisis et al., 1980.
1996.
1960.
3
5
9
Kawakami et al.,
Schauer et al .,1971.
DeVries and Binkley,
6
1986; determined for
Aisaka et al., 1991.
1972.
7
the recombinant
Uchida et al.,1984. 10Brunetti et al., 1962.
11
enzyme.
Sommer et al., in
prep.
of special interest, because the nucleophilic attack of an active site residue on 4-OH
is thought to represent an important step in catalysis (see below). The presence of a
free carboxyl and a glycosidic hydroxyl group is absolutely indispensible.
Already in the 1970s experiments were performed with amino acid-modifying
reagents to elucidate the role of special amino acids for catalysis (Table 4). First of
all, reaction of the enzyme with 14C-pyruvate followed by reduction with NaBH4
leads to the covalent linkage between the e-amino group of a lysine residue in the
active site and the carbonyl group at C2 of the substrate. The use of 14C-pyruvate
results in a radioactive label covalently attached to the enzyme protein, giving evidence for the formation of a Schiff base in the course of the reaction. Therefore,
acylneuraminate lyases are class I-aldolases in contrast to class II-aldolases that are
characterized by the dependence of their activity on the presence of metal ions.
(Rutter et al., 1968). This is in correspondence to the observation that sialic acid
aldolase activity is not inhibited by EDTA. The significance of a lysine in catalysis
is confirmed by mutagenesis experiments. A gradual decrease of enzyme activity
could be observed after exchange of the lysine against arginine, glutamine or alanine,
respectively. First measurements revealed no lyase activity of the alanine mutant.
Histidine was discussed to be another important residue, acting as a reversible
378
Schauer, Sommer, Kruger, van Unen, and Traving
Table 3. Substrate Specificity of Acylneuraminate Pyruvate-Lyases Relative Cleavage Rate [%]
Compared to the Optimal Substrate Neu5Ac (=100%)
Pig kidney
Neu5Ac
Neu4,5Ac2
Neu5,9Ac 2
4-epi-Neu5Ac
7-epi-Neu5Ac
8-epi-Neu5Ac
7,8-di-epi-Neu5Ac
4-deoxy-Neu5Ac
7-deoxy-Neu5Ac
8-deoxy-Neu5Ac
9-deoxy-Neu5Ac
Neu5Gc
N-benzyloxycarbonyl-Neu5Ac
N-formyl-Neu5Ac
N-monochloracetyl-Neu5Ac
N-monofluoracetyl-Neu5Ac
N-succinyl-Neu5Ac
Neu5Ac-methylester
Neu2en5Ac
1001
01 0–104
321, 454
Clostridium perfringens2
Escherichia coli3
100
22*$
100
05*
165
35
145
_5*
05
05
735
551, 656, 474
93, 657
+8
20
110
0l,4
106
28*
98
~1
0
* Competitive inhibitor of the enzyme; $ the relatively high cleavage rate may have been caused
by impurities with Neu5Ac (or by partial de-O-acetylation)
1
5
Schauer and Wember, 1996.
Schauer et al., 1987.
6
2
Brunetti et al., 1962.
Schauer et al., 1971.
7
3
Comb and Roseman, 1960.
Aisaka et al., 1991.
4
8
Sommer et al., in prep.
Faillard et al., 1969.
acceptor for the proton of 4-OH. This was concluded from the inhibitory effect of
5-diazonium-l-H-tetrazole and diethylpyrocarbonate on enzyme activity. The pHdependent photooxidation by Rose Bengal in the presence of light also points to an
involvement of histidine. The serine-modifying reagents phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate also abolish lyase activity and
from the inhibition by several agents like bromopyruvate or p-chloromercuribenzoate it can be seen that an SH-group might play a role in the reaction.
This can be further strengthened by the inhibitory effect of heavy metal ions like
Cu2+, Fe2+, Hg2+ and Ag2+.
The Catalytic Mechanism of Acylneuraminate Pyruvate-Lyases
Based on the results of the inhibition studies, Nees et al. (1976) proposed a
model for the lyase reaction, according to which the reaction starts with the formation of a Schiff base between the enzyme and the open chain substrate as described
above. Then, an unprotonated histidine abstracts a proton as a nucleophilic group
from the 4-OH of sialic acid. The bond between C3 and C4 is split and acylmannosamine is set free, while the carbanion of pyruvate is still linked to the lysine residue.
Finally, the proton taken up by the histidine is transferred to the carbanion and the
379
Acylneuraminate Pyruvate-Lyases
Table 4. Inhibition of Acylneuraminate Lyase Activity by Amino Acid-Modifying Reagents
Modifying agent
Modified group
Pyruvate
NaBH 4 + 14 C-pyruvate
Cyanide
5-Diazonium-1-H-tetrazole
Diethylpyrocarbonate
Rose Bengal + light
lodoacetamide
Bromopyruvate
p-Chloromercuribenzoate
5,5-Dithlobis-2-nitrobenzoic acid
N-Ethylmaleimide
o-Phenanthroline
Phthalaldehyde
PMSF
Diisopropylfluorophosphate
N-Bromosuccinimide
(NH 4 ) 2 SO 4
EDTA
DTT
Mercaptoethanol
*Competitive inhibition.
1
Nees et al., 1976.
2
Ferrero et al., 1996.
3
Schauer and Wember, 1996.
Schiff base
Lys
His
His
His
His, Cys, Met, Lys
SH
SH
SH
SH
SH, metal ions
SH next to NH2
Ser
Ser
Trp
C. perfringens
+ 1*
+ 1,7,8
+8
+ 1,8
+8
Pig kidney
+ 2.4.5*
+
4
+9
+
3,9
+ 3,9
+3
+ 4*
+
+4
+3
3
4
+
+
6
7,8
2,4,5
+
+7
_7 + 8
+ 3,9
+ 3.9
+2
_5
+3
+
3
+
3,9
+
9
5
+8
+ 4,5
+2
_ 4,5
7
SH-protection
SH-protection
7
5
8
_9
+2
–5
2
5
+
4
Aisaka et al., 1991.
Uchida et al., 1984.
6
Barnett et al., 1971.
E. coli
_9
–
DeVries and Binkley, 1972.
Schauer et al., 1971.
9
Sommer et al., in prep.
Schiff base is hydrolyzed releasing free pyruvate and the original enzyme molecule.
Baumann et al. (1989) showed by NMR-studies that the a-form of the substrate
molecule is reacting with the enzyme. Therefore, the authors proposed that the
closed ring form of Neu5Ac is bound by the enzyme and only then the pyranose
ring is opened to form the Schiff base (see below).
Elucidation of the three-dimensional structure of the lyase from E. coli mainly
confirmed these ideas (Izard et al., 1994). The enzyme monomer represents an a/Bbarrel structure consisting of eight alternating a-helices and B-sheets, which are Cterminally followed by three additional a-helices. The active site pocket is located
at the C-terminal end of the barrel. Apart from the critical lysine residue there could
be identified eight further residues to line the active site pocket. Interestingly, there
was no histidine in the right position to act as a proton acceptor for 4-OH, but a
tyrosine residue instead.
The alignment of the sequence data obtained from C. perfringens and the porcine lyase peptides with the other so far known lyase primary structures (Fig. 1)
showed the respective lysine and tyrosine residue to be absolutely conserved. The
same is true for some of the other residues that were found to be present at the
active site of the E. coli lyase. However, neither a histidine nor a cysteine residue is
conserved throughout all sequences, although both of them had been proposed to
be involved in catalysis due to the inhibition studies (see above). Additional interactions were noted to be present, e.g., hydrogen bonds between serine 47, threonine
380
Schauer, Sommer, Kruger, van Unen, and Traving
Fig. 1. Alignment of the major part of the acylneuraminate pyruvate-lyase amino acid sequences
of Clostridium perfringens, Escherichia coli, Haemophilus influenzae, Trichomonas vaginalis, mouse
and fetal human heart.
48 and tyrosine 137 of the E. coli sequence, respectively, and the carboxylate group
(Lawrence et al., 1997). Furthermore, there might be contact between aspartate 191
and glutamate 192 of the E. coli sequence, respectively, and the glycerol side chain
of the substrate (Lawrence et al., 1997).
The question for the identity of the proton acceptor for the 4-OH is so far still
not answered. It is also not yet clear whether the reaction starts by the nucleophilic
attack of the proton acceptor on 4-OH of the closed ring and only then the ring is
opened and the Schiff base formed, as is proposed by Zbiral et al. (1992) and
Schauer and Kamerling (1997), or whether out of the equilibrium between the closed
ring and open chain form the latter is reacting (Lawrence et al., 1997) with lysine
first to form the Schiff base, before the nucleophilic attack on C4-OH takes place.
We hope that we can contribute to the refinement of the present model of lyase
reaction by crystallization of both the clostridial and the porcine lyase, which is now
under way and by evaluation of the mutagenesis experiments with the microbial
enzyme.
Relationship of Acylneuraminate Pyruvate-Lyases
The homogenous size of the acylneuraminate pyruvate-lyase monomers is
reflected by the almost identical length of the microbial primary structures (Traving
Acylneuraminate Pyruvate-Lyases
381
et al., 1997). The high number of 86 amino acids was found to be conserved throughout all four microbial sequences. Calculations of the percentage of identical amino
acids between the four complete microbial lyase structures (Traving et al., 1997)
revealed the highest degree of similarity between the enzymes from the Gram-negative species H. influenzae and the protozoon T. vaginalis. Surprisingly, the similarity
between the lyases of H. influenzae and the other Gram-negative species E. coli is
only low. The clostridial enzyme stands in between. This unexpected pattern of lyase
relationship points to a possible horizontal transfer of microbial lyase genes that has
also been proposed for sialidases (Roggentin et al., 1993). It is also intriguing to ask
whether the genetic information for lyases originated in microbial species (Bacteria
or Archaea) and was then transferred to higher animals.
The comparison between the two partial mammalian EST-sequences showed
them to be almost identical (Fig. 1). Three of the porcine peptides were completely
identical to the other two mammalian sequences and one peptide sequence differs in
two positions to one or two of them, respectively. Thus, the mammalian lyases seem
to form an individual, highly related group (Sommer et al., 1997) apart from the
microbial enzymes. Accordingly, the similarity between the mammalian and the
microbial lyases is only low (Grobe et al., 1998), although the properties of the
porcine enzyme are similar to the bacterial lyases. These assumptions have to be
confirmed by complete sequencing of at least the porcine lyase gene. The almost
complete identity of the mammalian ESTs with the porcine peptide sequences as
well as the homology to the microbial primary structures confirms the identity of
the ESTs as partial lyase genes.
Recently, a subfamily of a, B-barrel proteins with homology to acylneuraminate
pyruvate-lyases was defined (Lawrence et al., 1997). Apart from these lyases, this
family comprises several enzymes and other proteins, which share a similar threedimensional structure and also have the ability to form Schiff bases, but catalyze
different reactions and thus have completely different biological functions.
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