From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
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ABSTRACT |
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The two hyaluronan synthases (HASs) from
Streptococcus pyogenes (spHAS) and Streptococcus
equisimilis (seHAS) were expressed in Escherichia
coli as recombinant proteins containing His6 tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their
stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239-4245). Kinetic characterization of the
enzymes in isolated membranes gave Km values for
UDP-GlcUA of 40 ± 4 µM for spHAS and 51 ± 5 µM for seHAS. In both cases, the
Vmax profiles at various concentrations of
UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In
contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal
behavior as the UDP-GlcNAc concentration was increased, with a Hill
number of ~2, indicating significant cooperativity. The Hill number
for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of
cooperativity for UDP-GlcNAc in this enzyme. The Km
values for UDP-GlcNAc were 60 ± 7 µM for seHAS and
149 ± 3 µM for spHAS in the isolated membranes. The
kinetic characteristics of the two affinity-purified HAS enzymes were
assessed in the presence of cardiolipin after 8-9 days of storage at
-80 °C without cardiolipin. With increasing storage time, the
enzymes showed a gradual increase in their Km values for both substrates and a decrease in
Vmax. Even in the presence of cardiolipin, the
detergent-solubilized, purified HASs had substantially higher
Km values for both substrates than the
membrane-bound enzymes. The KUDP-GlcUA for
purified spHAS and seHAS increased 2-4-fold. The
KUDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher Km values, the Vmax values for the purified HASs
were only ~50% lower than those for the membrane-bound enzymes.
Significantly, purified spHAS displayed the same cooperative
interaction with UDP-GlcNAc (nH ~ 2), whereas
purified seHAS showed no cooperativity.
HA1 is a polysaccharide
composed of two alternating sugars, The first HAS gene to be cloned was from Group A Streptococcus
pyogenes (14-16). When the bona fide HAS from Group C
Streptococcus equisimilis was later cloned (17), the seHAS
protein showed 70 and 72% identities to spHAS at the nucleotide and
amino acid sequence levels, respectively. After discovery of the spHAS
gene, a related family of homologous cDNAs and enzymes was then
found in eukaryotes (18, 19). These HASs include human HAS1 and HAS2
(20-22); murine HAS1, HAS2, and HAS3 (19, 23, 24); chicken HAS2 and
HAS3 (19); and Xenopus laevis HAS1, HAS2, HAS3, and HAS-related sequence (19, 25-29). In addition, HASs have also been
identified and cloned (30) from chlorella virus PBCV-1 (A98R) and from
Pasteurella multocida (31). Although the two streptococcal
HASs are very similar, the HAS from P. multocida is quite
different structurally. Similarities among the prokaryotic and
eukaryotic members of this HAS family have been reviewed (18). These
various HAS enzymes comprise a large family of proteins with many
common features and regions of amino acid sequence identity or similarity.
To understand the important role of the HA polysaccharide in normal
development and health and in various diseases, it is critical to know
more about the HASs, the enzymes responsible for HA synthesis. We need
to know how these HASs work to assemble the HA polymer and how the
enzymes are regulated. In the accompanying paper (32), we reported the
purification and lipid dependence of active recombinant spHAS and seHAS
expressed in Escherichia coli. In the present study, we have
determined, for the first time in the absence of other streptococcal
proteins or factors, the kinetic constants for both HAS enzymes in
membranes and after detergent solubilization and purification. A
preliminary report of these findings was reported earlier (33).
Materials, Strains, and Plasmids--
Reagents were from Sigma
unless stated otherwise. Media components were from Difco. S. pyogenes strain S43/192/4 and Group C S. equisimilis
strain D181 were from the Rockefeller University collection. E. coli SURETM cells were from Stratagene. The HAS open
reading frames from S. pyogenes (15, 16) and S. equisimilis (17) were inserted into the pKK223-3 vector (Amersham
Pharmacia Biotech) and cloned into E. coli
SURETM cells. The spHAS and seHAS proteins (16, 17)
contained a C-terminal fusion of 6 His residues. When a comparison was
made between seHAS-H6 in E. coli membranes and
seHAS in streptococcal membranes, there was no decrease in HAS
activity, normalized for HAS protein, due to the His6
fusion (data not shown).
Cell Growth and Membrane Preparation--
Membranes from
S. pyogenes and S. equisimilis were obtained by
modifications of a protoplast method (34), as reported previously (16).
Membranes from E. coli were isolated by a variation of the
protoplast method of Ito et al. (35). The SURETM
cells containing the construct were grown at 30 °C in Luria broth containing 50 mM glucose and trace elements (36) to an
A600 of 1.5 and then induced with
isopropyl- HAS Activity--
The standard determinations for HAS activity
were carried out in 100 µl of 25 mM sodium and potassium
phosphate, pH 7.0, containing 50 mM NaCl, 20 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
EGTA, 15-20% glycerol, 1 mM UDP-GlcUA (Fluka), and 0.8 µM UDP-[14C]GlcUA (267 mCi/mmol; NEN Life
Science Products). When assaying spHAS, 1.5 mM UDP-GlcNAc
was used, whereas 1.0 mM UDP-GlcNAc was used to assay
seHAS. n-Dodecyl Determination of Michaelis-Menten Constants
(Km)--
The Km values for both
UDP-GlcUA (KUDP-GlcUA) and UDP-GlcNAc
(KUDP-GlcNAc) were determined by varying one
substrate while holding the other constant. The incorporation of
substrate was monitored as described above. Assays were performed in
quadruplicate, and the Km values were determined by
either Lineweaver-Burk or Hill analysis (37). Protein concentrations
were determined with the Coomassie protein assay reagent (Pierce) using
bovine serum albumin as the standard (38).
Kinetic Characterization of Recombinant Membrane-bound spHAS and
seHAS--
The kinetic behaviors of the two recombinant HAS isozymes
in isolated E. coli membranes were compared using the paper
chromatography assay for incorporation of radiolabeled precursor sugar
nucleotides into HA (Figs. 1-4 and
Tables I and II). As noted previously in studies of HA chain elongation
(17), seHAS appeared to be 2-fold more catalytically active than spHAS.
The KUDP-GlcUA of both membrane-bound enzymes
differed only slightly, 40 ± 4 µM for spHAS (Table
I) and 51 ± 5 µM for
seHAS (Table II), at saturating
UDP-GlcNAc concentrations. The Vmax saturation
profiles, with respect to UDP-GlcUA utilization, for both enzymes in
E. coli membranes were hyperbolic (Figs. 1 and
2), and the Lineweaver-Burk plots
were linear (data not shown).
For both spHAS (Fig. 1B) and seHAS (Fig. 2B), the
specific enzyme activity at lower concentrations of UDP-GlcUA increased and then decreased as the concentration of UDP-GlcNAc increased. For
example, the spHAS enzyme was roughly twice as active in response to
the UDP-GlcUA concentration at 0.2 mM UDP-GlcNAc compared
with 2.0 mM UDP-GlcNAc (Fig. 1B). This biphasic
behavior probably reflects the partial competition of UDP-GlcNAc, at
high concentrations, for the UDP-GlcUA-binding site, although there is
no evidence that other sugar nucleotides are utilized by the
glycosyltransferase activities of either streptococcal HAS (16,
17).
Whereas the kinetic profiles of membrane-bound seHAS and spHAS for
UDP-GlcUA are similar, the kinetics of their interaction with
UDP-GlcNAc are not. The Vmax saturation profile
for spHAS with respect to UDP-GlcNAc utilization at various UDP-GlcUA
concentrations had a clearly sigmoidal shape (Fig.
3, A and B).
Interestingly, this sigmoidal behavior for UDP-GlcNAc utilization by
membrane-bound spHAS became more pronounced as the concentration of
UDP-GlcUA increased (Fig. 3B). When UDP-GlcNAc was varied at
a given concentration of UDP-GlcUA, the Hill analysis (Fig.
3C) revealed Hill numbers of ~2 (Table I). A Hill number
>1 indicates a degree of cooperativity associated with the spHAS
enzyme's ability to bind and utilize UDP-GlcNAc at fixed UDP-GlcUA
concentrations. Hill analysis gave a KUDP-GlcNAc
of 149 ± 3 µM for spHAS at saturating UDP-GlcUA concentrations.
The membrane-bound seHAS enzyme, however, did not respond to UDP-GlcNAc
in the same manner. Rather than showing a sigmoidal Vmax saturation profile with respect to
UDP-GlcNAc, seHAS showed a typical hyperbolic curve (Fig.
4). The Lineweaver-Burk analysis for
seHAS was linear and gave a KUDP-GlcNAc of
60 ± 7 µM at saturating UDP-GlcUA concentrations
(Table II). Hill plots for the seHAS interaction with UDP-GlcNAc gave
Hill numbers of ~1.0, indicating no cooperativity (Table II). As also
noted in Figs. 1B and 2B, when the UDP-GlcUA
concentration was increased, the specific activity of seHAS first
increased and then decreased (Fig. 4B). This biphasic behavior at low concentrations of UDP-GlcNAc could reflect the competitive interactions of UDP-GlcUA at the UDP-GlcNAc-binding site.
Kinetic Characterization of Purified Streptococcal HASs--
The
purification, storage, and stability of seHAS and spHAS and the effects
of CL on their activities have been described in the accompanying paper
(32). The kinetic characterization of both purified enzymes was
performed after 8-9 days of storage at
The kinetic characteristics observed for the pure enzymes were very
similar to those seen in Figs. 1-4 for the membrane-bound HASs. Both
spHAS and seHAS showed hyperbolic responses to UDP-GlcUA in the
presence of UDP-GlcNAc. Most notably, purified spHAS showed sigmoidal
behavior for its utilization of UDP-GlcNAc (Fig.
5A), but the response of seHAS
to UDP-GlcNAc was hyperbolic, not sigmoidal (data not shown).
Therefore, purified spHAS retained this distinct characteristic of
cooperativity displayed by the membrane-bound enzyme. The
Michaelis-Menten constants for utilization of both sugar nucleotides by
the purified enzymes were significantly increased when compared with
the membrane-bound enzymes. The saturation profiles, however, remained
very similar. For spHAS, KUDP-GlcUA and
KUDP-GlcNAc were 40 and 149 µM for
the membrane-bound enzyme, but increased to 175 and 434 µM,
respectively, for purified spHAS (Table
III). Likewise, for seHAS,
KUDP-GlcUA and
KUDP-GlcNAc were 51 and 60 µM for
the membrane-bound enzyme, but increased to 274 and 251 µM, respectively, for purified seHAS (Table
IV).
Although the Km values for the two substrates
increased 4-10-fold in the purified HASs, the overall catalytic
efficiencies of the two enzymes were very similar to those of the
membrane-bound enzymes. Based on their specific activities at
saturation and their abundance in the E. coli membranes used
for the purification (32), we estimate that the
Vmax for the pure HASs, after 8-9 days of
storage, is at least 50% of the Vmax for the
membrane-bound enzymes.
Another difference noted in the purified compared with the
membrane-bound enzymes is that both purified HASs were more sensitive to substrate inhibition, especially spHAS (Fig. 5). This increase in
the degree of inhibition with increasing sugar nucleotide concentration was apparent when either substrate was present at or over saturating concentrations. At low concentrations of the first sugar nucleotide, spHAS showed a plateau and then a decrease in velocity as the second
sugar nucleotide concentration increased. The
Vmax for spHAS increased and then dramatically
decreased as the UDP-GlcNAc concentration was varied from 5 µM to 2 mM at fixed concentrations of
UDP-GlcUA (Fig. 5A). Likewise, the same inhibition pattern occurred when the concentration of UDP-GlcUA was varied from 5 µM to 2 mM at fixed concentrations of
UDP-GlcNAc (Fig. 5B). This dramatic inhibition of the
purified HASs at high sugar nucleotide concentrations was not as
apparent in the membrane-bound species. These results indicate that the
recognition of the normal substrates is affected by other sugar
nucleotides and that "cross-talk" occurs between the two
sugar-binding sites and both substrates.
Historically, the first cell-free studies of HA biosynthesis used
Group A streptococcal bacteria. The pioneering work of Dorfman and
co-workers (39, 44) in the 1950s and 1960s showed that the
streptococcal HAS was located in the cell membrane, required Mg2+ ions, and used the two sugar nucleotide substrates
UDP-GlcUA and UDP-GlcNAc to polymerize a HA chain. Subsequently,
however, these and many other workers were unable to solubilize the
enzyme in an active and stable form or to purify it. Similarly,
eukaryotic HASs have not yet been purified. Isolation of the Group A
and Group C streptococcal HAS genes has now allowed us to express these
proteins in large amounts in E. coli (32).
All of the known enzymes catalyze reactions that use one or two (or,
rarely, three) substrates and produce one or two products. HASs are
unique among the enzymes characterized to date. The HAS has two
different enzyme activities (i.e. glycosyltransferases) in
the same protein, and the HA product after each sugar addition becomes
the acceptor for the next sugar addition. HASs are also membrane
enzymes. The overall reaction for the synthesis of one HA disaccharide
unit is shown in Equation 1,
INTRODUCTION
Top
Abstract
Introduction
References
1,3-linked glucuronic acid and
1,4-linked N-acetylglucosamine (1). Although the
structure of HA seems quite simple, the molecule, nonetheless, has
unusual physical properties that are important for its numerous
biological functions (2-5). For example, HA forms very viscous
solutions and gels due to its high molecular mass and its ability to
bind cations and to hydrate large amounts of water. This characteristic
of HA provides the viscous lubrication of synovial fluid and helps
provide cartilage with its viscoelasticity. These characteristics are
also ideal for the role HA has in the extracellular matrices (4, 5) of
the skin and virtually every vertebrate tissue as well as in the fluid
of the vitreous humor of the eye. HA also plays an important role in
morphogenesis, wound healing (6-9), and angiogenesis (10, 11). HA
receptors and HA-binding proteins, particularly CD44 (12) and the
receptor for hyaluronic acid-mediated mobility (RHAMM; Ref. 13),
modulate cellular responses to HA.
EXPERIMENTAL PROCEDURES
-D-thiogalactoside and grown for an additional
3 h. The cells were harvested by centrifugation at 4 °C for 30 min at 3000 × g, washed twice with phosphate-buffered saline containing 10% glycerol, and then frozen at
80 °C. The cell pellet was thawed and resuspended to 1% of the original culture volume in 20% sucrose, 30 mM Tris, pH 8.2, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin. Lysozyme (4 mg/ml) in 0.1 M EDTA, pH 8 (0.1% of the initial culture volume), was added, and the suspension was incubated for 40 min on ice with constant
mixing. Phenylmethanesulfonyl fluoride was added to a concentration of
46 µg/ml, and the suspension was sonicated three times for 30 s
each at 20 watts with a microtip (Model W-380, Heat Systems Ultrasonic,
Inc.). MgCl2 (60 mM) and DNase and RNase (1 µg/ml each) were added to the indicated final concentrations. After
20 min on ice with constant mixing, debris was removed by centrifugation (10,000 × g, 30 min, 4 °C). The
lysate was diluted with 1 volume of ice-cold phosphate-buffered saline
containing 10% glycerol, 1 mM dithiothreitol, and the
above protease inhibitors, and the membranes were harvested at
100,000 × g for 1 h. The membrane pellet was
washed once with phosphate-buffered saline containing 10% glycerol, 1 mM dithiothreitol, and the above protease inhibitors by
resuspension and centrifugation. The final pellet was stored frozen at
80 °C.
-D-maltoside (0.98 mM) and 2 mM CL were also present in assays
with the purified HAS, which was purified as described in the
accompanying paper (32). To initiate the enzyme reaction, 3 µg of
whole membrane protein or 0.3-0.5 µg of pure HAS was added, and the
mixtures were gently mixed in a Micromixer (Taitec) at 30 °C for
1 h. Reactions were terminated by the addition of SDS to 2% (w/v)
final concentration at room temperature. Incorporation of
[14C]GlcUA into high molecular mass HA was measured by
descending paper chromatography using Whatman No. 3MM paper developed
in 1 M ammonium acetate, pH 5.5, and ethanol (7:13). After
cutting out the origins, the amount of radioactivity present was
assessed using 1 ml of H2O and 5 ml of Ultimagold
scintillation fluid (Packard) and a Packard Model A2300 scintillation counter.
RESULTS
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Fig. 1.
Effect of UDP-GlcUA concentration on the
activity of membrane-bound spHAS at various concentrations of
UDP-GlcNAc. A, membranes containing recombinant
spHAS were incubated with the indicated concentrations of UDP-GlcUA
(UDP-GlcA) and 0.1 mM ( ), 0.2 mM
(
), 0.5 mM (
), 1 mM (
), 1.5 mM (
), or 2.0 mM (
) UDP-GlcNAc to assess
HAS activity as described under "Experimental Procedures." The
saturation profiles of all curves were hyperbolic and gave linear
Lineweaver-Burk plots (data not shown). B, a blowup of the
initial concentration range in A shows a decrease in
activity at low concentrations of UDP-GlcUA as the concentration of
UDP-GlcNAc increases.
Michaelis-Menten constants for spHAS in E. coli membranes
Michaelis-Menten constants for seHAS in E. coli membranes
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Fig. 2.
Effect of UDP-GlcUA concentration on the
activity of membrane-bound seHAS at various concentrations of
UDP-GlcNAc. A, membranes containing recombinant
seHAS were incubated with the indicated concentrations of UDP-GlcUA
(UDP-GlcA) and 0.01 mM ( ), 0.05 mM (
), 0.1 mM (
), 0.5 mM
(
), 1.0 mM (
), or 1.5 mM (
)
UDP-GlcNAc, and HAS activity was measured as described under
"Experimental Procedures." The saturation profiles of all curves
were hyperbolic and gave linear Lineweaver-Burk plots (data not shown).
B, a blowup of the initial concentration range of the
saturation profile in A shows an increase and then a
decrease in activity at low concentrations of UDP-GlcUA as the
concentration of UDP-GlcNAc increases.
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Fig. 3.
Effect of UDP-GlcNAc concentration on the
activity of recombinant membrane-bound spHAS at various concentrations
of UDP-GlcUA. A, membranes containing recombinant spHAS
were incubated with the indicated concentrations of UDP-GlcNAc and 0.0 1 mM ( ), 0.05 mM (
), 0.1 mM
(
), 0.5 mM (
), 1.0 mM (
), or 1.5 mM (
) UDP-GlcUA (UDP-GlcA). The saturation
profiles of all the curves showed sigmoidal behavior with respect to
UDP-GlcNAc at various UDP-GlcUA concentrations. B, a blowup
of the low concentration range of UDP-GlcNAc in A shows an
increase in the sigmoidal behavior of the enzyme toward UDP-GlcNAc as
the UDP-GlcUA concentration is increased. C, the Hill
analysis of the data from A yielded numbers that are
approximately equal to 2, thus indicating cooperativity associated with
the ability of spHAS to utilize UDP-GlcNAc in the presence of
UDP-GlcUA.
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Fig. 4.
Effect of UDP-GlcNAc concentration on the
activity of recombinant membrane-bound seHAS at various concentrations
of UDP-GlcUA. A, membranes containing recombinant seHAS
were incubated with the indicated concentrations of UDP-GlcNAc and 0.01 mM ( ), 0.05 mM (
), 0.1 mM
(
), 0.5 mM (
), or 1.0 mM (
) UDP-GlcUA
(UDP-GlcA). The saturation profiles of all the curves were
hyperbolic and gave linear Lineweaver-Burk plots (data not shown).
B, a blowup of the initial concentration range in
A shows a decrease in enzyme activity at low concentrations
of UDP-GlcNAc as the UDP-GlcUA concentration increases.
80 °C in the absence of
CL. The activities of the two affinity-purified synthases were
relatively stable from about day 6 to at least 4 weeks under these
conditions. Bovine CL was present at 2 mM in all the
kinetic assays since both enzymes are lipid-dependent and
highly stimulated by CL.
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Fig. 5.
Effect of UDP-GlcNAc and UDP-GlcUA
concentrations on the activity of purified spHAS. A,
dependence of the spHAS reaction rate on UDP-GlcNAc concentration.
Purified spHAS was stored without CL for 8 days and then assayed for
HAS activity, as described under "Experimental Procedures," in the
presence of 2 mM CL. Samples contained the indicated
concentrations of UDP-GlcNAc and 0.01 mM ( ), 0.05 mM (
), 0.1 mM (
), 0.5 mM
(
), 1.0 mM (
), or 2.0 mM (
) UDP-GlcUA
(UDP-GlcA). The saturation profiles of all the curves showed
sigmoidal behavior with respect to UDP-GlcNAc at various UDP-GlcUA
concentrations. The Hill analysis (Table III) of the data yielded
numbers that are approximately equal to 2. Thus, purified spHAS behaves
cooperatively with respect to UDP-GlcNAc utilization in the presence of
UDP-GlcUA. B, dependence of the spHAS reaction rate on
UDP-GlcUA. The enzyme was assayed as described for A with
the indicated concentrations of UDP-GlcUA and 0. 1 mM
(
), 0.2 mM (
), 0.5 mM (
), 1.0 mM (
), 1.5 mM (
), or 2.0 mM
(
) UDP-GlcNAc. The saturation profiles of all curves were
hyperbolic and gave linear Lineweaver-Burk plots (data not
shown).
Michaelis-Menten constants for purified spHAS after storage
80 °C in the absence of CL for 8 days
and then assayed in the presence of 2 mM bovine CL as
described under "Experimental Procedures" and in the accompanying
paper (32). In the absence of added CL, the values for
KUDP-GlcU and KUDP-GlcNAc were
62 ± 7.8 and 1360 ± 128 µM, respectively, and
Vmax was 4.6 ± 0.4 nmol/µg/h.
Michaelis-Menten constants for purified seHAS after storage
80 °C in the absence of CL for 9 days
and then assayed in the presence of 2 mM bovine CL as
described under "Experimental Procedures" and in the accompanying
paper (32). In the absence of added CL, the values for
KUDP-GlcUA and KUDP-GlcNAc were
54 ± 7.9 µM and 1.06 ± 0.15 mM,
respectively, and Vmax was 13.6 ± 1.1 nmol/µg/h.
DISCUSSION
where n is the number of disaccharide units. Although
it seems straightforward, the enzyme must possess at least six (and probably seven) different functions to perform this overall
reaction, as shown in Fig. 6.
Numerous questions about the details and mechanism of this complex
reaction can be answered now that the streptococcal HAS enzymes have
been purified.
(Eq. 1)
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Fig. 6.
Multiple enzyme functions needed for HA
biosynthesis. The diagram shows membrane-bound HAS and the six
independent activities required for any HAS to make a disaccharide unit
and to extend the growing HA chain. The enzyme has two distinct sugar
nucleotide-binding sites, two glycosyltransferase activities, at least
one (and perhaps two) acceptor-binding sites for HA, and a HA transfer
function. Before it is released, the chain can grow to a length of
40,000 monosaccharides or more, corresponding to a mass of >8 × 106 Da. The sugar nucleotide substrates are produced and
used by the synthase inside the cell, and the HA chain is continuously
transferred (translocated) so that it is extruded into the exterior of
the cell to form a capsule or to be secreted into the medium. The
dashed lines represent the cross-talk hypothesis (described
under "Results") in which a sugar nucleotide, even one other than a
normal substrate, transiently occupies, and thereby competes for, the
other sugar nucleotide-binding site. UDP-GlcA,
UDP-GlcUA.
HAS enzymes are unique in an additional respect because the two sugar nucleotide substrates are structurally so similar, they each have the possibility of competing with the other for the appropriate UDP-sugar-binding site on the enzyme. This cross-talk hypothesis is illustrated in Fig. 6 by the dashed lines that show, for example, UDP-GlcUA interacting with the UDP-GlcNAc-binding site. Although the HASs do not misincorporate other sugar nucleotides into the growing HA chain, other UDP-sugars may transiently occupy a binding site and thus be competitive inhibitors. Initial experiments have indicated that 0-1 mM concentrations of sugar nucleotides like UDP-Glc or UDP-GalUA or even UDP alone decrease the rate of HA synthesis by spHAS or seHAS.2 With high concentrations of one or both of the correct substrates, we observed enzyme inhibition in the presence of a third sugar nucleotide. Even with just the two normal substrates, the rate of HA synthesis becomes biphasic if the concentration of one UDP-sugar is much greater than the other (e.g. Fig. 2A at a ratio of 100:1). There are no previous reports of this cross-talk phenomenon affecting HA biosynthesis. Observation of this kinetic behavior reflects the advantage of studying purified HAS free from other sugar nucleotide-binding proteins or glycosyltransferases.
The scheme in Fig. 6 does not indicate whether sugars are added to the
growing HA chain individually or in a coordinated manner as a
disaccharide unit as proposed by Saxena et al. (40) for -glycosyltransferases predicted to contain two functional domains by
hydrophobic cluster analysis. Our present kinetic data for the
streptococcal HASs do not allow discrimination between these two
models. The model of Saxena et al. (40) predicts that HA synthesis occurs by addition to the reducing end, as suggested by Prehm
(41). However, this model is not consistent with recent reports that HA
synthesis (42) and Type 3 polysaccharide synthesis, mediated by the
related synthase from Streptococcus pneumoniae (43), occur
from the nonreducing end of the growing polysaccharide chains.
Definitive answers regarding the direction of polysaccharide chain
growth for the HASs and related synthases may require the identification, perhaps utilizing mass spectrometry, of putative UDP-polymer intermediates with the UDP attached at the reducing end.
Stoolmiller and Dorfman (44) reported the apparent KUDP-GlcUA and KUDP-GlcNAc as 50 µM and 0.5 mM, respectively, in isolated S. pyogenes membranes. Their UDP-GlcUA kinetic results gave a linear Lineweaver-Burk plot. They noted, however, that the kinetics of UDP-GlcNAc utilization did not behave in the same linear manner when plotted in double-reciprocal form. These investigators were therefore the first to note sigmoidal behavior of spHAS in streptococcal membranes in response to increasing UDP-GlcNAc concentration. van de Rijn and Drake (34) reported, for detergent-solubilized spHAS, values of 39 µM for KUDP-GlcUA and 150 µM for KUDP-GlcNAc, but did not detect a sigmoidal response of the velocity saturation profile when UDP-GlcNAc was varied. They also reported the specific activity of spHAS as 19.4 nmol of UDP-GlcUA/h/mg of extracted membrane protein. We have reported in the accompanying paper (32) that the specific activities of purified spHAS and seHAS are 5,500 and 12,000 nmol/h/mg, respectively. These values are consistent with spHAS composing only ~0.3% of the membrane protein in S. pyogenes cells (17). The addition of bovine CL substantially increased the specific activity of purified spHAS and seHAS, respectively, to 20,000 and 35,000 nmol/h/mg. The catalytic constants for purified spHAS and seHAS in the presence of bovine CL at 30 °C were, respectively, 22 and 36 monosaccharides/s. These values are in close agreement with those reported for seHAS assayed at 37 °C in whole membranes (17).
The above apparent Michaelis-Menten values for the two substrates were
determined using either crude streptococcal membranes or
detergent-solubilized membrane extracts, both of which contain other
sugar nucleotide-binding proteins and glycosyltransferases and which
could contain potential regulatory factors for the HAS enzyme. Here,
for the first time, we have characterized the kinetic behavior of
purified spHAS and seHAS and these enzymes in membranes containing no
other streptococcal proteins. The results demonstrate that the
n-dodecyl -D-maltoside-solubilized, purified
enzymes behave very similarly to the membrane-bound enzymes. This is an important finding because many studies have reported that HAS activity is irreversibly lost upon solubilization of the
protein in a wide variety of nonionic detergents (45). The
KUDP-GlcUA values for membrane-bound
spHAS and seHAS were 40 and 51 µM, respectively. The
KUDP-GlcNAc values for membrane-bound spHAS and
seHAS were 149 and 60 µM, respectively.
Detergent-solubilized, purified HASs showed essentially the same
kinetic characteristics as the membrane-bound enzymes, with the
exception of their Km values. The
KUDP-GlcUA values increased ~4-fold for
purified spHAS and seHAS, although the latter enzyme was not saturated
even at 1.5 mM. Both enzymes also displayed increased
Km values for UDP-GlcNAc after purification; the
KUDP-GlcNAc values increased 4-fold for spHAS
and 5-fold for seHAS. We also noted that upon storage at 80 °C in
the absence of CL, these enzymes slowly lost activity
(t1/2 ~ 2-3 months), and the biggest change
appeared to be in KUDP-GlcNAc, which got
progressively larger with time of storage. The stimulation of either
HAS by CL is due to a large decrease in
KUDP-GlcNAc and an increase in
Vmax (32).
Based on our recent findings, the sensitivity of HASs to detergent
solubilization can now be explained. Radiation inactivation analysis
revealed that the active spHAS and seHAS species are monomers of the
HAS protein in complex with ~16 CL molecules (46). This conclusion is
supported by the results in the accompanying paper (32) showing that
the activity of affinity-purified HAS, which has been depleted of CL,
is very low. The HAS enzymes are highly lipid-dependent and
are most effectively stimulated by CL. Preliminary mass spectroscopic
analysis indicated that even when purified in the absence of exogenous
CL, the enzymes still contain residual associated CL. Therefore, the
likely reason for why most detergents inactivate HAS (47-49) is that
these detergents displace the CL required for enzyme activity. Even
with CL present, most nonionic detergents will compete more efficiently
than CL for interaction with the protein. The mild detergent
n-dodecyl -D-maltoside is apparently strong
enough to solubilize HAS from membranes, but not so strong that the
enzyme is stripped of CL. Identification of n-dodecyl
-D-maltoside as a useful detergent for
solubilizing HAS was a substantial contribution (45).
Two substantial differences are apparent between the two enzymes. First, the seHAS enzyme is intrinsically about twice as active as spHAS. This was apparent in this and the accompanying study (32) with both membrane-bound and purified enzymes and in an earlier study (17) that examined the rates of HA chain elongation by gel filtration analysis. Since the Group C HA capsule is typically larger than the Group A capsule, this difference could be due to the Vmax between the two HASs. Second, spHAS, but not seHAS, is complexly regulated by UDP-GlcNAc. The spHAS interaction with this substrate shows a cooperative activation of the enzyme as the UDP-GlcNAc concentration increases. This sigmoidal behavior indicates that spHAS has a second binding site for UDP-GlcNAc that is involved in regulation rather than catalysis. Such allosteric-like regulation is usually observed in enzymes that function as oligomers, not enzymes that are active as a monomeric species such as HAS.
For a bacterium to synthesize a HA capsule, three different genes must usually be present. These genes, which encode three different enzymes, are arranged in an operon designated the HA synthesis (or has) operon (50, 51). Two of the enzymes are needed for the cell to produce large amounts of the two UDP-sugar precursors, and the third enzyme is HAS, encoded by the gene hasA. UDP-Glc dehydrogenase (whose gene is designated hasB) is required to make UDP-GlcUA from UDP-Glc in an oxidation reaction that utilizes 2 mol of NAD+/mol of UDP-Glc. UDP-Glc pyrophosphorylase (the hasC gene) creates UDP-Glc from UTP and Glc-1-P. Since UDP-Glc is the precursor from which many of the other sugar nucleotides are made, the amount of UDP-Glc produced by a cell will regulate the total amount of all the cell's sugar nucleotides. Bacteria like Group A Streptococcus that make HA capsules usually have two different genes for this pyrophosphorylase enzyme to increase greatly the total amount of cellular sugar nucleotides and thereby to support synthesis of the large amount of HA in the extracellular capsule.
If the bacterial cells did not greatly expand their sugar nucleotide pool, the very active HAS would make HA and deplete the cell of UDP-GlcUA and UDP-GlcNAc. Since the latter is also needed for cell wall synthesis, such depletion would stop the cell from growing. This, in fact, occurs when a streptococcal HAS gene is expressed in another bacterial species that produces the two substrates, but lacks the additional enzyme (hasC) needed to enlarge the sugar nucleotide pools (14-16). These cells do not grow well.
The cooperative kinetic response of spHAS to UDP-GlcNAc may reflect an
evolutionary adaptation by Group A Streptococcus to ensure
that cell growth is not impaired by the production of the HA capsule.
Because spHAS activity responds to changes in UDP-GlcNAc concentration
in a sigmoidal manner, the enzyme cannot attain its
Vmax until this concentration is very high. This
kinetic regulation of spHAS may ensure that cell wall synthesis does
not compete with capsule production for UDP-GlcNAc. It is unclear why
the seHAS enzyme is not similarly regulated by UDP-GlcNAc or if this lack of regulation is, in fact, a reason why Group A organisms are more
pathogenic in humans than Group C strains. Availability of purified
streptococcal HASs will facilitate future studies on their role in
bacterial virulence and the mechanisms by which these enzymes perform
the multiple functions required for HA biosynthesis.
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ACKNOWLEDGEMENTS |
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We thank Anil Singh for technical support and Debbie Blevins for help preparing the manuscript.
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FOOTNOTES |
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* This work was supported by NIGMS Grant GM35978 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 405-271-1288;
Fax: 405-271-3092; E-mail: paul-weigel{at}ouhsc.edu.
The abbreviations used are: HA, hyaluronan or hyaluronic acid; HAS, hyaluronan synthase; seHAS, S. equisimilis hyaluronan synthase; spHAS, S. pyogenes hyaluronan synthase; CL, cardiolipin.
2 K. Kumari, V. L. Tlapak-Simmons, and P. H. Weigel, manuscript in preparation.
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REFERENCES |
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