From the Children's Hospital Oakland Research
Institute, Oakland, California 94609, the § Department of
Biochemistry and Molecular Genetics and the ¶ Center for AIDS
Research Molecular Biology Core Facility, University of Alabama,
Birmingham, Alabama 35294
Received for publication, May 21, 2002, and in revised form, November 13, 2002
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ABSTRACT |
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Streptococcus pneumoniae
hyaluronate lyase is a surface antigen of this Gram-positive human
bacterial pathogen. The primary function of this enzyme is the
degradation of hyaluronan, which is a major component of the
extracellular matrix of the tissues of vertebrates and of some
bacteria. The enzyme degrades its substrate through a Hyaluronan (HA)1 is a
glycan that is abundantly present in nearly all vertebrate tissues,
especially the extracellular matrix, and in some bacteria such as
Streptococcus zooepidemicus. It is a polymeric substance
built from a repeating disaccharide units of hyaluronic acid with the
chemical formula [-elimination
process called proton acceptance and donation. The inherent part of
this degradation is a processive mode of action of the enzyme degrading
hyaluronan into unsaturated disaccharide hyaluronic acid blocks from
the reducing to the nonreducing end of the polymer following the
initial random endolytic binding to the substrate. The final
degradation product is the unsaturated disaccharide hyaluronic acid.
The residues of the enzyme that are involved in various aspects of such
degradation were identified based on the three-dimensional structures
of the native enzyme and its complexes with hyaluronan substrates of
various lengths. The catalytic residues were identified to be
Asn349, His399, and Tyr408. The
residues responsible for the release of the product of the reaction
were identified as Glu388, Asp398, and
Thr400, and they were termed negative patch. The
hydrophobic residues Trp291, Trp292, and
Phe343 were found to be responsible for the precise
positioning of the substrate for enzyme catalysis and named hydrophobic
patch. The comparison of the specific activities and kinetic properties
of the wild type and the mutant enzymes involving the hydrophobic patch
residues W292A, F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V allowed for the characterization of every mutant and for the
correlation of the activity and kinetic properties of the enzyme with
its structure as well as the mechanism of catalysis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4)GlcUA(
1
3)GlcNAc(
1
]n (Fig. 1) (1). Hyaluronan isolated from
natural sources has an enormous size, up to 25,000 disaccharide units
or 107 Da. The polymer interacts with water to create a
strikingly viscoelastic solution. These unique mechanical properties
are utilized in, for example, joints as a shock absorber (2). In
addition to the mechanical properties, hyaluronan synthesis and
degradation is finely regulated to allow initiation of other
biochemical processes. For example, hyaluronan is involved in multiple
signal transduction processes (3, 4), often utilizing other
macromolecules for interactions such as CD44 or RHAM (5, 6).
Through these molecules or these transduction processes, HA influences
many essential processes, for example, cell migration and
development.
View larger version (17K):
[in a new window]
Fig. 1.
Structure of polymeric hyaluronan. The
1,4 linkage connecting the hyaluronic acid disaccharide units is
degraded by S. pneumoniae and other bacterial hyaluronate
lyase enzymes.
Exogenous elements, such as bacteria including members of the
Streptococcus species, degrade the hyaluronan of its host
organism, including humans, through the action of hyaluronate lyase
enzymes by the process of -elimination (7-9). In contrast, the
endogenous degradation of hyaluronan is performed by the hyaluronidase
enzymes of the host that utilize a mechanistically distinct hydrolysis mechanism (7). The exact molecular mechanism of the lyase action was
largely unknown until relatively recently when the first structural information on bacterial hyaluronate lyase enzymes was obtained by
means of x-ray crystallography (10). Similar structural information for
the endogenous hyaluronidases is not available at present except for
that of the homologous bee venom enzyme (11). Therefore, the details of
this process are still unclear and are based on comparison with other
hydrolytic polysaccharide degrading enzymes.
Hyaluronate lyase from Streptococcus pneumoniae, a
Gram-positive human pathogen (12-14), has recently been cloned,
overexpressed, and purified (15, 16). The availability of large
quantities of the protein led to extensive biochemical and biophysical
characterization of the enzyme together with its crystallization (17)
and three-dimensional crystal structure determination (10). Structure
determination of the native form of the enzyme was followed by
molecular modeling and characterization of crystal-based structures of
enzyme-substrate complexes using di-, tetra-, and hexasaccharide units
of hyaluronan (18, 19) (Fig. 2). Finally,
a crystal structure of hyaluronate lyase from another
Streptococcus species, Streptococcus agalactiae, was also elucidated in its native and complex forms with the
disaccharide unit of HA degradation (20, 21). The three-dimensional
x-ray crystal structures of hyaluronate lyases show the enzyme as a globular protein built from at least two distinct domains: a helical -domain and a
-sheet
-domain (Fig. 2). The
-domain is
traversed by an elongated deep cleft where the HA substrate binds and
where it is degraded to disaccharides.
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The comparison of both structure groups, from S. pneumoniae and S. agalactiae, allowed for additional conclusions and generalization of the proposed mechanism of action of the lyase (22, 23). The catalytic residues (Asn349, His399, and Tyr408), as well as the residues of the hydrophobic (Trp291, Trp292, and Phe343) and the negative (Glu388, Asp398, and Thr400) patches that were implicated in the activity of the lyase were clearly identified and analyzed based on approaches similar to those used in the structural studies described above (see Fig. 3) (10, 19, 23). These studies include the structures of the native S. pneumoniae and S. agalactiae hyaluronate lyase, the structures of complexes with the substrates or products (disaccharide product of degradation, tetra- and hexasaccharide hyaluronan units), and site-directed mutation studies used in conjunction with kinetic studies of the corresponding mutant enzyme forms (22, 24).
The enzymes with mutated catalytic residues Asn349,
His399, and Tyr408 were generated and isolated
previously, and their enzymatic properties, including specific
activities along with kinetic parameters, were determined and analyzed
as described (23). The correlation between the activity of the mutant
enzymes, kinetic properties of the mutant enzymes, and their structures
clearly confirmed the Asn349, His399, and
Tyr408 residues as being directly involved in the
catalysis. A detailed catalytic mechanism of -elimination based
degradation of HA, termed proton acceptance and donation (PAD), was
proposed (see Figs. 3 and 4). The proposed mechanism involves five
distinctive steps: (i) binding to the negatively charged hyaluronan
substrate to the enzyme binding cleft with precise positioning of the
substrate being directed by the hydrophobic patch consisting of
residues Trp291, Trp292, and
Phe343; (ii) catalysis involving primarily the catalytic
residues Asn349, His399, and Tyr408
with the resulting cleavage of the glycosidic
1,4 bond and
generation of the disaccharide product; (iii) proton (hydrogen)
exchange between the corresponding His399 and
Tyr408 residues with the water microenvironment, a step
that readies the enzyme for the next round of catalysis; (iv) release
of the disaccharide product utilizing the negative patch composed of the Glu388, Asp398, and Thr400
residues; and finally (v) translocation of the remaining polymeric hyaluronan substrate by 1 disaccharide unit toward the reducing end of
the substrate (see Figs. 3 and 4) (10, 19, 23).
Here we report further analysis of the properties of this enzyme by
mutating the residues of the hydrophobic patch and providing the
analysis of their role and importance in catalysis. Additional insights
into this catalytic mechanism are supplied by the investigations of the
specific activities and the kinetic properties of the wild type and
mutant forms of this lyase.
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EXPERIMENTAL PROCEDURES |
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Materials-- Hyaluronan used in this study was from human umbilical cord (Sigma; sodium salt, Lot 11K1517). All other chemicals were purchased from either Fisher or Sigma.
Cloning of the Mutant Form of the Enzyme--
Plasmid DNA
preparations were obtained using the QIAprep kit from Qiagen, Inc.
Mutagenesis was performed using the QuikChange kit and procedure of
Stratagene. The template for the single mutations, W292A and F343V, was
plasmid pET-SpHyal-His6, pMJJ004 (15), containing a truncated but active S. pneumoniae hyaluronate
lyase (Ala168-Glu891 of the mature enzyme).
Multiple mutations were made progressively by performing mutagenesis
using templates and primers with the desired combination of mutations.
Mutants were identified by automated DNA sequence analysis. The
following primers, with the desired changes indicated in bold italic
type, were used in the mutagenesis procedures (+ and denote the
upstream and the downstream primers, respectively): W291A+,
GAGCATTGTTGGGAACGCTGCAGATTATGAAATCGG; W291A
,
CCGATTTCATAATCTGCAGCGTTCCCAACAATGCTC; W292A+,
CATTGTTGGGAACTGGGCAGATTATGAAATCGGTACAC; W292A
,
GTGTACCGATTTCATAATCTGCCCAGTTCCCAACAATG; F343V+, GACGACTGATAACCCAGTAAAGGCTCTAGGTGGAAAC; and F343V
,
GTTTCCACCTAGAGCCTTTACTGGGTTATCAGTCGTC. The resultant clones
were used to transform Escherichia coli BL21 (DE3) cells
using standard procedures (25).
Production of Enzyme Forms-- The recombinant wild type S. pneumoniae hyaluronate lyase enzyme from E. coli was overexpressed and purified as previously described (10, 15, 17). The mutant forms of the enzyme: (i) single mutants W292A and F343V; (ii) double mutants W291A/W292A and W292A/F343V; and (iii) one triple mutant W291A/W292A/F343V used in this study were overexpressed and purified in the same fashion as the wild type enzyme. The wild type and the mutant enzymes were stored in 10 mM Tris-HCl (pH 7.4), 2 mM EDTA, 150 mM NaCl, and 1 mM dithiothreitol buffer at 5 mg/ml protein concentration for further use. The activity unit for the enzyme was defined as the molar amount of the enzyme that produces 1 µM product/min (15). The presence of the mutations in the produced proteins was additionally identified (in addition to DNA sequence-based analysis) by mass spectrometry experiments as described under "Other Methods."
Initial Velocity and Specific Activity Measurement and Data
Analysis--
During the process of enzyme-catalyzed degradation of
polymeric hyaluronan, a double bond in the glucuronic moiety is
introduced between carbon atoms C-4 and C-5. This bond formation in the
product of HA degradation induces a spectral shift with the absorption maximum at ~232 nm. This absorption was followed experimentally to
detect changes in the progress of the reaction for the wild type and
the mutant enzymes. The HA degradation reaction was carried out in
quartz cuvettes containing 300 µl of a reaction buffer with the HA
substrate. The reaction buffer was 50 mM sodium acetate, 10 mM CaCl2 at pH 6.0. The reaction was initiated
by the addition of 10 µl of enzyme in the same buffer to the mixture
of buffer and the specified amounts of the substrate. The amounts of
the enzyme in the reaction mixture were modified as needed because of
different abilities to degrade HA, and they were as follows: wild type,
10 ng; F343V, 33 ng; W292A, 500 ng; and W292A/F343V, 4800 ng. The
measurement was initiated exactly 15 s after the enzyme was added
to the reaction mixture. The progress of the reaction of HA degradation
was followed by detecting the absorbance at 232 nm. The spectral
measurements were performed using the BioSpec-1601PC UV-visible
spectrophotometer (Shimadzu) equipped with a thermoelectrically
temperature controlled cell holder to perform the reaction when
temperatures were raised above room temperature. The limits for the
detection of the specific activity were directly related to the
sensitivity of the spectrophotometer used, which was the absorbance at
232 nm of 0.001 or specific activity of 0.01 unit/min. Enzymes with
smaller activity were considered inactive. All of the materials used in
this study were preincubated at 37 °C using a water bath, and all of
the subsequent procedures were also performed at this temperature.
Concentration determinations used a molar absorption coefficient for
the product of HA degradation of 5.5 × 103
liter/mol1 cm
1 as described (15, 26).
For the specific activity measurements, the enzyme was added to a cuvette containing 300 µl of a 0.2 mg/ml of polymeric HA in the reaction buffer. To investigate the influence of the hydrophobic effect versus hydrogen bonding on activity, the specific activity measurements were also performed with the addition of 150 mM NaCl in the reaction buffer. The product absorbance was measured every 15 s for 6 min. The specific activity was calculated using UV-1601 PC Kinetics software (Shimadzu) and previously reported methodologies (10, 15).
For the initial velocity measurements the polymeric hyaluronan substrate concentration ranged from 0.012 to 2 mM. The moles of HA were expressed as moles of its disaccharide unit based on its molecular weight. The measurements were performed similarly to the initial velocity measurements with the exception that the data points were recorded in 6-s intervals for 1.5 min. The initial velocity for the degradation reaction was calculated based on the increase of absorbance during the first 90 s of the reaction.
The measured data for initial velocity, vi, and
the varied concentrations of substrate, S, were fit to the
Michaelis-Menten equation, v = VmaxS/(Km + S), with a nonlinear regression program (Scientist
Micromath). In this equation, the Km is the
Michaelis constant, and Vmax is the maximum
velocity. For all of the experiments, goodness-of-fit statistics showed
that R-squared and correlation values were greater than
0.957 and 0.982, respectively. As the results of the curve
fitting show, the program afforded values of
Vmax and Km for the enzyme
forms analyzed as well as their respective standard deviations ().
From these curve-fitted data the values of
Vmax/Km parameter, and the
95% confidence limits (3x
) were obtained.
Determination of the Size of the Degradation Product-- Hyaluronan was degraded as previously described (10). Briefly, for 1000 µl of 2.0 mg/ml of hyaluronan solution in 50 mM sodium acetate, 10 mM CaCl2 at pH 6.0, the following amounts of the enzyme forms were used: wild type, 35 ng; F343V, 2.5 µg; W292A, 2.5 µg; W292A/F343V, 50 µg; W291A/W292A, 50 µg; and W291A/W292A/F343V, 50 µg. The mixture was incubated at 37 °C and collected at 1, 5, and 10 min and 3, 7, 20, and 24 h (100 µl each). The degradation product mixtures were immediately separated on a Superdex peptide HR 10/30 column (Amersham Biosciences) using 10 mM ammonium acetate buffer at pH 7.4. The eluting peak fraction identities were confirmed by electrospray mass spectrometer on a Micromass Quattro LCZ tandem mass spectrometer using atmospheric pressure ionization and the conditions previously reported (10, 27).
Crystallization of Mutants and Data Collection-- The crystallization for the mutant forms of the enzyme was performed similarly to the crystallization of the wild type hyaluronate lyase and as described (17). A hanging drop vapor diffusion with VDX culture plates and siliconized glass cover slides were used. Briefly, equal volumes (~1 µl each) were mixed of reservoir solution and mutant proteins W292A, F343V, W292A/F343V, W291A/W292A, and W291A/W292A/F343V in buffer described earlier and at protein concentrations of 6.2, 5.5, 5.1, 5.6, and 5.10 mg/ml, respectively. The reservoir solution contained 60-65% saturated ammonium sulfate, 0.2 M sodium chloride, 2% dioxane, and 50 mM sodium citrate buffer at pH 6.0.
The crystals of the inactive mutant proteins W291A/W292A and W291A/W292A/F343V were in addition soaked in a hexasaccharide hyaluronan substrate (HA6) for 10 h prior to cryo-freezing. The soaking solution contained 75% saturated ammonium sulfate, 10 mM sodium citrate buffer at pH 6.0 and 10 mM HA6. All of the mutant crystals were cryo-protected in 30% xylitol (w/v), 80% saturated ammonium sulfate, and 10 mM sodium citrate buffer, pH 6.0, and flash frozen in liquid nitrogen before diffraction data collection.
The diffraction data collection was performed at a synchrotron source utilizing Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory beamline 5.0.1. The x-ray wavelength was 1.0 Å, and the crystal diffraction was recorded on a Quantum 4u CCD detector using the oscillation method. The data were integrated and scaled using the HKL2000 package (28). The unit cells of crystals of mutants and their complexes were isomorphous to the native ones. The final data processing parameters are reported in Table I.
Determination of Structures of Mutants and Their
Complexes--
The native SpnHL crystal structure without water
molecules (10) was used as the model for the mutant and complex
structure solution. The Rfree flag was assigned
to 1% of reflection for W292A, F343V, and W292A/F343V mutant
diffraction data sets and 2% for W291A/W292A and W291A/W292A/F343V
mutant complexes with HA6 data sets to validate the
refinement progress (29, 30). Initially, a round of rigid body
refinements using only the model structure was performed using the
refmac5 maximum likelihood protocol (31, 32). The mutated residues were
modified manually using the program O (33). The refinements were
continued using the restrained and unrestrained protocols of refmac5
(31, 32) and were traced using inspection on graphics with the program O (33). The structures were refined against all reflections from 20.0 Å to the highest resolution available without any (F) cut-off (see Table I). The electron density for the HA6
substrate for the W291A/W292A and W291A/W292A/F343V complex structures
was clearly identified and was followed by the incorporation of the HA6 substrates into this density as previously described
(19, 34, 35). The topologies/parameter files for the substrate were
manually created following our earlier studies to reflect ideal
stereochemical values. Additional refinements including individual
anisotropic B-factor refinements for all structures, inspection, and
manipulation of structures on graphics using O together with
incorporation of water molecules placed into 3
peaks in sigma-A
weighted Fo
Fc
maps following standard criteria were performed. After further
refinements, water molecules whose positions were not supported by
electron density, at 1
contouring, in sigma-A weighted
2Fo
Fc maps were
deleted. A variety of stereochemical (36) and other analyses (33, 37)
were periodically performed to locate possible model errors (38). The
number of water molecules incorporated and the final refinement
parameters are reported in Table I.
Other Methods-- The enzyme concentration was determined by the UV absorption at 280 nm using the molar extinction coefficient calculated based on the native or mutant S. pneumoniae hyaluronate lyase amino acid residue sequence data (15, 39). The calculated molar extinction coefficients were 127,090 for the native enzyme and the F343V mutant, 121,590 for the W292A and W292A/F343V mutants, and 116,090 for the W291A/W292A and W291A/W292A/F343V mutants.
Mass spectrometry experiments to confirm the presence of mutations were
collected on an LCQ quadrupole ion trap (ThermoFinnigan) mass
spectrometer equipped with an electron spray source operating in
positive ion mode. Raw data were deconvoluted using BioMass deconvolution algorithm in the Xcalibur BioWorks software package from
ThermoFinnigan. The structural figures were prepared with Ribbons (40)
and O (33).
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RESULTS |
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Construction of the Mutant Forms of the Enzyme-- A plasmid containing a partial (Ala168-Glu891) but active S. pneumoniae hyaluronate lyase, pET-Sphyal-His6 (pMJJ004) (15), was used as the template to create the single mutants SpHyal-W292A (pMJJ020) and SpHyal-F343V (pMJJ021) by site-directed mutagenesis. These two mutants then served as templates with the primers encoding W291A and W292A, respectively, to create the double mutants SpHyal-W291A/W292A (pMJJ022) and SpHyal-W292A/F343V (pMJJ023). To make the triple mutant, SpHyal-W291A/W292A/F343V (pMJJ024), the SpHyal-W292A/F343V clone served as the template with the mutagenesis primer encoding W292A. The generated clones were used to transform the E. coli overexpressing cells BL21 (DE3) (25).
Overexpression and Purification of the Wild Type and Mutant
Enzymes--
The recombinant wild type S. pneumoniae
hyaluronate lyase protein from E. coli was obtained as
previously described (10, 15). The mutant forms of the enzyme, W292A,
F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V, were
overexpressed and purified following the same procedure as that for the
wild type enzyme (15) or other mutant forms reported previously (10).
Briefly, the overexpression was performed by growing E. coli
BL21 (DE3), harboring the appropriate clone, in LB medium with
ampicillin selection and
isopropyl-thio--D-galactopyranoside (1 mM)
induction. The purification procedure consisted of enzyme isolation
from a cell lysate using a chelating Sepharose fast flow nickel
affinity chromatographic step (Novagen) followed by a size exclusion
column on Superdex 75 (Amersham Biosciences) and a high resolution
anion exchanger using a MonoQ column (Amersham Biosciences).
The presence of mutations was confirmed by mass spectrometry experiments using electrospray ionization of the protein samples. The results yielded molecular masses for W292A, F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V of 83,208, 83,273, 83,097, 83,102, and 83,027 Da, respectively. These masses correspond to the calculated molecular masses: 83,173, 83,240, 83,058, 83,125, and 83,010 Da, respectively. The mutations were also confirmed by obtaining their three-dimensional structures (see below).
To confirm the maintenance of the overall fold of the produced mutant enzymes, they were crystallized. The crystallization experiments yielded crystals in essentially the same conditions and of the same habit as the native enzyme crystals previously reported by us (10, 17). To conform this information and to confirm the structural fold in the active site of the enzyme together with the presence of the expected mutated residues, for all mutants three-dimensional crystal structures were obtained (Table I). The structures showed no significant differences with the native enzyme in their overall fold as well as in the fold in the catalytic cleft (Figs. 3 and 4). The mutated residues were identified in the electron densities as expected. Therefore, the observed changes in activity are due to the changes of amino acid residues introduced by site-directed mutagenesis.
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Initial Velocity Experiments with a Hexasaccharide Hyaluronan
Substrate--
The kinetic parameters of the wild type and mutant
enzymes are interpreted in the context of the PAD mechanism (Fig.
5) including: (i) substrate binding, (ii)
catalysis, (iii) proton exchange with microenvironment, (iv) product
release, and (v) translocation of remaining HA. The fifth step, the
translocation of remaining HA, accounts for the processive character of
the enzyme as shown previously (41). Also, the product release step may
account for the nearly irreversible nature of this catalysis.
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In our earlier mutation and kinetic studies that were designed to
investigate the involvement of the PAD mechanism in pneumococcal lyase
catalysis, we derived an equation using the method of net rate
constants (42) to describe the initial velocity of the enzyme,
i (23). The effects described by this equation show that
for the enzyme catalyzed reaction, a relationship between measured
kinetic parameters and functional interpretation can be defined: (i)
changes only in Vmax/Km for
various mutants indicate that the enzyme binds the substrate with a
different affinity, (ii) changes only in Vmax
indicate that the nascent product/substrate translocation proceed at a
different rate, and (iii) changes in both
Vmax/Km and
Vmax show that the mutation in the enzyme
affects some combination of the binding, the catalysis, the release of
product, and the translocation steps. Although the
Km may approximate the substrate binding affinity,
this parameter can also be affected by the other rate and equilibrium
constants during the catalysis, and therefore in addition to the
Km parameter,
Vmax/Km is used as a
descriptive parameter for hyaluronate lyase catalyzed reaction
(23).2
Kinetic Properties of the Mutant Forms of the Enzyme-- The wild type enzyme obeyed Michaelis-Menten kinetics. The values of Vmax and Km for degradation of the hyaluronan substrate were 0.23 ± 0.01 mmol/(min × mg) and 0.09 ± 0.03 mM, respectively (Table II). The availability of the three-dimensional crystal structures for the wild type hyaluronate lyases from the Streptococcus species and the complexes with substrates/products provided a unique opportunity to correlate the kinetic and structural properties of this enzyme. The mutations of the catalytic residues H399A, N349A, and Y408F have already been investigated, and all have significantly reduced values of Vmax and Vmax/Km as compared with the wild type enzyme (23). In the current study we report the analysis of the residues of the hydrophobic patch residues Trp291, Trp292, and Phe343. Based on the three-dimensional structure analysis, the region of hydrophobic patch occupies ~5% of the total cleft area, and the hydrophobic character in this region of the enzyme is significantly higher than that of the surrounding areas (10, 18, 19). As implied in the proposed PAD mechanism, these amino acid residues should be responsible for the precise positioning of the polymeric substrate for catalysis. The analysis of the structural environment in the catalytic cleft also suggested that these residues might be responsible for the size of the generated product of HA degradation (10). Five mutants were produced, W292A, F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V, to investigate the properties of the hydrophobic patch of the enzyme. Of these mutants, the W291A/W292A and W291A/W292A/F343V forms were totally inactive; therefore, no specific activities or kinetic parameters were determined. The remaining three mutant forms, W292A, F343V, and W292A/F343V, were fully analyzed, and the specific activities or kinetic parameters were determined as described under "Experimental Procedures" (Table II). The specific activities for all three partially active mutants were significantly compromised when compared with the wild type enzyme. Similarly the Vmax values for each of the characterized mutants were much lower. The Km values for the W292A and W292A/F343V mutants were significantly lower that that of the wild type. The Km value for the F343V mutant was comparable with that of the wild type (Table II). The changes observed in Km for W292A and W292A/F343V mutants, but not for the F343V mutant are likely caused by the small alterations in the positioning of the substrate in the cleft and the resultant change in the binding of substrate. The analysis of the structure of the native enzyme (10), its complexes with the substrate (19, 34), and the mutant enzymes reported here suggests that the F343V mutation alone likely results in the misalignment of the substrate along the clef and therefore likely decreased binding. Similar changes in the alignment along the cleft axis and the resultant change in Km were observed in our earlier work for the R243V mutant (10, 34).
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Based on the structural information available, the longest HA unit that
entirely fits in the catalytic cleft of the enzyme is the
hexasaccharide. The hexasaccharide is built from three disaccharide
units named HA1, HA2, and HA3, which correspond to the units
originating from the reducing end toward the nonreducing end of this
polymer. The side chain of Trp292 interacts through a
hydrophobic interaction with the sugar rings of the HA2 disaccharide of
the substrate, primarily with the
N-acetyl--D-glucosamine group (Fig. 3).
Similarly, the Phe343 side chain interacts with the
hydrophobic ring moieties of HA1, primarily with the
N-acetyl-
-D-glucosamine group. Although the interactions of Phe343 with the substrate are significantly
weaker that that of Trp292, they are still pronounced
(longer distances and smaller number of them). Trp291
interacts primarily through a hydrogen bonding interaction network with
the N-acetyl-
-D-glucosamine group of HA1. The
Vmax parameter is compromised for all three
partially active mutant enzymes, suggesting a direct influence on the
catalytic process, product release, and/or translocation and
involvement of each of the affected residues in such process. The role
of Phe343 seems to be primarily related to catalysis and
less in the binding of substrate. Based on the kinetic data, the
Trp291 and Trp292 residues seem to be involved
in both the binding as well as the precise positioning of the substrate
for the catalytic process (34).
The mutations W291A/W292A and W291A/W292A/F343V have completely inactivated the enzyme, preventing the determination of kinetic parameters that are used to characterize these mutants (Table II). According to the structural data of the enzyme complexes with tetra- and hexasaccharides (19), the Trp292 residue interactions are more significant that those of the remaining residues of the hydrophobic patch, and therefore mutating this residue has a more detrimental influence on the activity, especially when combined with changes to other residues of the hydrophobic patch. All of the information presented in the analysis of the wild type and the mutant enzymes, their specific activities, and kinetic properties is consistent with and confirms our previous structural and mechanistic studies of the enzyme (10, 18, 19, 23).
Size of the Degradation Product--
The wild type and all mutant
enzymes were tested with respect to the size of the product of
degradation. All of the active enzyme forms showed similar profiles of
hyaluronan degradation with the final product of degradation identified
as the unsaturated hyaluronic acid disaccharide,
2-acetamido-2-deoxy-3-O-(-D-gluco-4-enepyranosyluronic acid)-D-glucose. With short degradation times (~1 min), a
small population of tetra- and hexasaccharides products was present for
the three characterized mutants and the wild type enzyme (Fig. 6). The inactive mutants (specific
activity below the instrumental detection limit of 0.01 unit/min) were
not tested, because they did not degrade the substrate.
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Crystal Structures of the Mutants and Their Complexes--
The
protein components of all three crystal structures of the mutants
(W292A, F343V, and W292A/F343V) and the two complexes of the two
additional mutants with HA6 (W291A/W292A and
W291A/W292A/F343V) are nearly identical to one another as well as to
the structure of the native enzyme (10). The detailed description of
the native structure and its complexes with hyaluronan have been
reported elsewhere (10, 18, 19). The small structural changes present in the catalytic cleft and in the area of the hydrophobic residues are
limited to the differences specifically related to the missing hydrophobic side chains of the mutated residues only. For the inactive
mutants W291A/W292A and W291A/W292A/F343V a hexasaccharide hyaluronan
substrate is present in the catalytic cleft. The position and the
orientation of the HA6 substrate are similar to those in
the structure reported earlier (19, 34). The substrate conformation,
including the carbohydrate ring structures, is also similar to the
structures reported earlier (19, 34, 35). The lack of hydrophobic
residues in mutants did not significantly change the position of the
substrate but caused, as expected, its small distortion out of the
ideal position in the binding cleft with respect to the catalytic
residues and therefore displacement from ideal position for catalysis
(Figs. 3b and 4).
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DISCUSSION |
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Structural Properties of the Enzyme--
The structure of
4hyaluronate lyase enzyme from S. pneumoniae shows a two
domain enzyme with a catalytic -domain at the N terminus with a
structure of
5/
5 barrel with two layers
of
-helices, five on the inside of the barrel and five on the
outside (Fig. 2) (10). At one end of such a barrel, the helices and
loops between them form an elongated, deep cleft characteristic of
glycan-binding enzymes. Structural and site-directed mutagenesis
studies proved that hyaluronan substrate binds in this cleft and is
also degraded at this location. In addition to the
-domain, there is
an additional
-sheet domain located toward the C terminus of the
enzyme. This domain extends further to facilitate the enzyme binding to
the cross-bridges of the peptidoglycan structures of pneumococci. The
-sheets, in general, are arranged into a
-sandwich structure. The
-domain likely facilitates the access of the substrate to the
catalytic cleft by covering up or opening the space over the cleft (10,
19).
The cleft region spanning the -domain is of appropriate size to
accommodate binding of the polymeric hyaluronan, is positively charged,
and has only a small number of interactions with the
-sheet domain.
These interactions are limited only to the selected residues, mainly
Asn580, at the edge of the
-domain that is facing the
cleft and the
-domain (Fig. 2). The negatively charged hyaluronan
substrate was found to bind to the cleft. The positive charges in the
cleft that complement the negative charge of the substrate are due to the accumulation of lysines and arginines that appear to be highly conserved among different bacterial species expressing this enzyme (10). The active site residues are grouped together at one side of the
cleft and are composed of three distinct groups of residues: (i) the
catalytic group composed of His399, Tyr408, and
Asn349, (ii) a hydrophobic (aromatic) patch composed of
Trp291, Trp292, and Phe343, and
finally (iii) a negative patch composed of Glu388,
Asp398, and Thr400 (10, 19, 23) (Fig. 3). The
hydrophobic patch function appears to involve the selection of cleavage
sites on the substrate chain for catalysis and the precise positioning
of the substrate for cleavage by the catalytic group of residues. The
catalytic group of residues is responsible for the cleavage of the
glycosidic
1,4 linkage between HA1 and HA2 chains of the substrate
through a five-step PAD catalytic mechanism as described below (10). The structural studies showed that the side chain of catalytic Asn349 forms hydrogen bonds with the carboxyl group of
glucuronate moiety of the HA1 disaccharide and acts as an electron
sink. This in turn causes acidification of the C-5 hydrogen of the C-5
carbon of the same glucuronate. A base, the His399 residue,
then withdraws and accepts this more acidic hydrogen, resulting in the
rehybridization of the C-5 carbon of the glucuronate. Simultaneously,
the third catalytic residue, Tyr408, acts as an acid and
donates its phenolic proton (hydrogen) to the glycosidic oxygen of the
1,4 bond to be cleaved. Protonation of this oxygen induces bond
cleavage, subsequent formation of a C-4-C-5 double bond of the same
glucuronate, and release of the unsaturated disaccharide product from
the cleft. The remaining hyaluronan substrate is translocated toward
the reducing end direction of the chain so that the penultimate
disaccharide may then interact with the three catalytic residues (Fig.
5). During the process the enzyme donates the acquired proton on
His399 to the surrounding water molecules and attracts one
proton to Tyr408. In this manner the enzyme is prepared for
the next round of catalysis.
Selection of the Residues for Mutations--
In our previous study
we have studied the catalytic mutants of the enzyme N349A, H399A, and
Y408F as well as two mutants modifying the binding of the enzyme to the
substrates R243V and N580A (10, 23). For the current study, we selected
residues of the hydrophobic patch Trp291,
Trp292, and Phe343 for the site-directed
mutations. Based on the structural studies, their suggested role in the
substrate degradation was to select the 1,4 glycosidic bond for
degradation and position it precisely with respect to the catalytic
group residues. Also, a suggestion has been raised that the hydrophobic
patch residues are essential for the determination of the size of the
product of degradation, i.e. disaccharide instead of tetra-
or hexasaccharide, and for the positioning of the hyaluronan substrate
in the enzyme cleft for catalysis. The precise positioning of the
hyaluronan substrate is primarily accomplished by the hydrophobic
interactions of carbohydrate rings of the substrate and the
hydrophobic/aromatic side chains of the residues selected for the
mutation studies, primarily Trp292 and Phe343.
To eliminate this interaction to study its effect on the enzyme activity and the size of the degradation product, the substitution of
these residues to smallest amino acids like Ala or Val was preferred.
The Trp291 residue interacts with the substrate mostly
through the hydrogen bonding of its NE1 nitrogen (Fig. 3a)
(34) unlike the ring-ring hydrophobic interaction of the substrate with
Trp292 and Phe343 (Table I). Therefore, this
residue was not selected for single mutation. The double and triple
mutants were generated to study the additive effects of such changes on
the enzymatic activity and the size of the product of degradation.
Kinetic Properties of the Mutant Forms of the Enzyme--
All five
of the mutants involving the hydrophobic residues in the cleft of
hyaluronate lyase were expected to reduce the values for both
Vmax and
Vmax/Km. For example, the
W291A mutation removes Trp291, and therefore the indole
nitrogen of Trp291, which otherwise would participate in
several hydrogen bonds with the
N-acetyl--D-glucosamine moiety of HA1 of the
reducing end of the disaccharide to be removed, is not available to
participate in substrate binding or positioning (19). Thus
Trp291 would appear to serve an important role in the
catalytic mechanism of this enzyme because it acts to finely position
the substrate for catalysis and therefore directly assists in the
catalytic process. The W292A mutation voids the enzyme from
establishing the key hydrophobic interaction of Trp292 with
the N-acetyl-
-D-glucosamine moiety of the HA2
disaccharide. The kinetic analysis indicating disruption to enzymatic
function is also supported by the structural studies that indicate the importance of this hydrophobic interaction between the substrate and
the enzyme in substrate positioning for catalysis. The two Trp amino
acid residues cooperatively interact with the substrate on both sides
of the
1,4 glycosidic linkage; this interaction and the
corresponding enzyme-catalyzed reaction are disrupted by mutating
either or both residues. Site-directed mutagenesis of
Trp292 in the W292A mutant causes the enzyme to lose 98%
of its activity and compromises the binding of the substrate as
reflected in the Km and
Vmax/Km parameters (Table
II). Even though the single mutant of Trp291 was not
produced, its influence on enzyme activity is expected to be even more
detrimental than that of the Trp292 mutant alone. The
double mutant produced, W291A/W292A, abolished the enzymatic activity
totally, clearly illustrating the importance of both Trp residues in
enzyme catalysis. Finally, the mutation of Phe343 in the
F343V mutant has a significant role in activity, because the mutant
enzyme preserves only 70% of its original activity. Even though the
substrate binding characteristics of this mutant do not appear to be
significantly affected, as approximated by a Km
value of 0.08 ± 0.01 mM (comparable with that of the
wild type enzyme (0.09 ± 0.03 mM)), the
Vmax and
Vmax/Km values are
significantly smaller (Table II). The Km parameter might also be influenced by other rate or equilibrium constants. The
availability of the structural information, especially that of a
complex of HA with the enzyme (19), suggests that perhaps the binding
changes are not too significant for this mutant as compared with the
wild type enzyme. In principle, the F343V mutation, however, may
influence both the binding and the translocation of the substrate.
Alternatively, the mutant enzyme may perform catalysis and product
release or both at different rates. In this specific case the changes
in the catalysis and product release or both are more likely.
The combined mutants of any two of these residues or all three of them render the enzyme essentially inactive. For the W291A/W292A double mutant and the W291A/W292A/F343V triple mutant, no activity could be detected even while utilizing the excess of the enzyme and increasing the time course for the reaction to several days. The only double mutant with some residual activity was W292A/F343V, which retained only 0.08% activity of the wild type enzyme. The values of Vmax or Vmax/Km were drastically smaller, whereas the Km was determined to be 3.82 ± 0.36 mM. All three parameters showed an enzyme drastically compromised in its catalytic abilities. It is evident that the function of the hydrophobic patch residues, Trp291, Trp292, and Phe343, is very important for the catalytic process of the enzyme. The individual mutations of these residues impact the activity to a very significant degree, and the double mutants obtained render the enzyme either inactive or essentially inactive.
The mutations of the noncatalytic, hydrophobic patch residues of the
hyaluronate lyase were designed to test the different roles of these
residues, particularly those involving positioning of the substrate for
catalysis and in substrate binding. None of the residues analyzed
participate in the catalytic function of this enzyme. These conclusions
are based on the intrinsic chemical properties of these residues as
well as their positioning relative to the substrate molecule observed
in the three-dimensional structures of each of the corresponding
substrate-enzyme complexes (19). All of these residues are part of the
catalytic -domain, and they are well separated from the
-sheet
domain of the enzyme, which controls the access to the cleft.
Therefore, these mutations provide insight into role of the catalytic
domain and about the reaction catalyzed by the enzyme.
Selection Process of the Size of the Degradation Product--
The
size of the final degradation product for the wild type and the active
mutant enzymes is the C-4-C-5 unsaturated disaccharide of hyaluronic
acid. None of the mutants produced products different from that of the
wild type (data not shown). At very short degradation times, all of the
enzymes were shown to produce tetra- and hexasaccharides; however, with
time, they were all eventually degraded to disaccharide units (data not
shown). These data do not support earlier suggestions that the
hydrophobic patch residues play a direct role in the determination of
the size of the degradation product (10). However, the location of
these three hydrophobic residues in the cleft appear to be crucial in
defining the precise positioning of the substrate on both sides of the
glycosidic bond to be degraded as well as the processive and efficient
nature of the enzymatic degradation of all consecutive 1,4 linkages
(to be degraded). In this manner primarily disaccharides are generated
by the enzyme because the
1,4 linkages separate distinct
disaccharide units of HA (Fig. 1). Production of small amounts of
tetra- and hexasaccharide units of HA instead of only disaccharides
might be a result of random errors in the selection process of the
linkage to be degraded. The presence of either Trp292 or
Phe343 or even the lack of these two residues (W292A/F343V
mutant) seems to protect the selection process for substrate
degradation. However, the specific activity of the mutants causes the
process to slow considerably, because the mutant residues are not
expected to optimally position the substrate for catalytic degradation.
The catalysis proceeds even with some of these residues not being available to position the substrate perfectly for catalysis, and every
1,4 linkage is still degraded, albeit less efficiently.
The determination of specific activities for the mutant enzymes was also performed at higher salt concentration (ionic strength) to test the relation of hydrophobic versus, for example, hydrogen bonding effects (Table II). At higher ionic strength one might expect that the hydrophobic effect would be the major component as compared with the hydrogen bonding network. The activities clearly show a slight decrease as the ionic strength rises, but the changes are not very significant. These results are, however, consistent with the assumption that hydrophobic forces are very important and are essential for catalysis.
Structures of the Mutants and Their Complexes with
Substrate--
The structures of three mutants W292A, F343V, and
W292A/F343V and two mutants complexes with the HA6
substrate, W291A/W292A and W291A/W292A/F343V, fully support the
conclusions made above. The mutagenesis of hydrophobic residues did not
modify significantly, if at all, the positions of the mutated residues
but depraved the substrate of this enzyme of hydrophobic interactions
made possible by the selected residues. The kinetic measurements
performed of the mutants reflect primarily the changes caused by the
mutation changes in the side chains and their interactions. These
changes were shown to be very important for the enzyme-catalyzed
reaction (Figs. 3b and 4 and Table II). The lack of
hydrophobic interaction with the substrate did not drastically misplace
the substrate; the substrate, as shown in the two complex structures
reported here, is only slightly misplaced as compared with the earlier structural information (19, 34). However, the small modifications to
the position of the substrate, especially with respect to the catalytic
residues, Asn349, His399, and
Trp408, are detrimental for catalysis. The precise
positioning of the substrate by the hydrophobic residues present in the
cleft of the enzyme, Trp291, Trp292, and
Phe343 through the hydrophobic interaction with the
substrate, including interactions of hydrophobic sugar rings of
hyaluronan with hydrophobic planar moieties of Trp292 and
Phe343, are absolutely essential for full activity of the enzyme.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Songlin Li, Ewa Witkowska, and Stephen J. Kelly for help and assistance. The clones of the mutant forms of the enzyme were obtained at University of Alabama at Birmingham Molecular Biology Core Facility and were sequenced at the DNA Sequence Core Facility. The mass spectrometry experiments for identification of molecular masses of mutants were performed at Stanford University Mass Spectrometry facility. The diffraction data were collected at the Berkeley Center for Structural Biology, Advanced Light Source, Lawrence Berkeley National Laboratory using Beamline 5.0.1.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI 44078 (to M. J. J.).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.
The atomic coordinates and the structure factors (code 1N7N, 1N7O, 1N7P, 1N7Q, and 1N7R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Children's
Hospital Oakland Research Inst., 5700 Martin Luther King Jr. Way,
Oakland, CA 94609. Tel.: 510-450-7932; Fax: 510-450-7910;
E-mail: MJedrzejas@chori.org.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M204999200
2 K. B. Taylor, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: HA, polymeric hyaluronan; GlcUA, D-glucuronic acid; HA6, hexasaccharide unit of hyaluronan; PAD, proton acceptance and donation.
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