The active sites of DspB and other family 20 hexosaminidases possess three highly conserved acidic residues, several aromatic residues and an arginine at subsite1.. From these results, w
Trang 1biofilm-releasing b-hexosaminidase from a periodontal
pathogen, in substrate hydrolysis
Suba G A Manuel, Chandran Ragunath, Hameetha B R Sait, Era A Izano, Jeffrey B Kaplan and Narayanan Ramasubbu
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA
The biofilm attachment and detachment properties of
the oral pathogen Aggregatibacter
actinomycetemcomi-tans (formerly Actinobacillus actinomycetemcomitans)
are mediated by a soluble b-N-acetylglucosaminidase
(dispersin B abbreviated to DspB; EC 3.2.1.52) [1]
Interestingly, DspB exhibits biofilm-detachment activ-ity not only for A actinomycetemcomitans, but also for biofilms produced by several bacterial species including Gram-positive species (Staphylococcus epide-rmidis) [2] and Gram-negative species (Actinobacillus
Keywords
biofilm detachment; dispersin B; enzyme
mechanism; hydrolysis; site-directed
mutagenesis
Correspondence
N Ramasubbu, Department of Oral Biology,
C-634, MSB, UMDNJ, 185 South Orange
Ave, Newark NJ 07103, USA
Fax: +1 973 972 0045
Tel: +1 973 972 0704
E-mail: ramasun1@umdnj.edu
(Received 28 August 2007, revised 30
September 2007, accepted 1 October 2007)
doi:10.1111/j.1742-4658.2007.06121.x
Dispersin B (DspB), a family 20 b-hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves b(1,6)-linked N-acetylglu-cosamine polymer In order to understand the substrate specificity of DspB, we have undertaken to characterize several conserved and noncon-served residues in the vicinity of the active site The active sites of DspB and other family 20 hexosaminidases possess three highly conserved acidic residues, several aromatic residues and an arginine at subsite)1 These res-idues were mutated using site-directed mutagenesis and characterized for their enzyme activity Our results show that a highly conserved acid pair in b-hexosaminidases D183 and E184, and E332 play a critical role in the hydrolysis of the substrates pH activity profile analysis showed a shift to a higher pH (6.8) in the optimal activity for the E184Q mutant, suggesting that this residue might act as the acid⁄ base catalyst The reduction in kcat observed for Y187A and Y278A mutants suggests that the Y187 residue (unique to DspB) located on a loop might play a role in substrate specific-ity and be a part of subsite +1, whereas the hydrogen-bond interaction between Y278A and the N-acetyl group might help to stabilize the transi-tion state Mutatransi-tion of W237 and W330 residues abolished hydrolytic activity completely suggesting that alteration at these positions might col-lapse the binding pocket for the N-acetyl group Mutation of the conserved R27 residue (to R27A or R27K) also caused significant reduction in kcat suggesting that R27 might be involved in stabilization of the transition state From these results, we conclude that in DspB, and possibly in other structurally similar family 20 hydrolases, some residues at the active site assist in orienting the N-acetyl group to participate in the substrate-assisted mechanism, whereas other residues such as R27 and E332 assist in holding the terminal N-acetylglucosamine during the hydrolysis
Abbreviations
DspB, dispersin B; Hex A, human b-hexosaminidase; MuGlcNAc, 4-methylumbelliferyl-b- D -N-Acetylglucosamine; PGA, homopolymer of b(1,6)-linked GlcNAc; pNPGlcNAc, p-nitrophenyl-b- D -N-acetylglucosamine; SpHex, S plicatus hexosaminidase.
Trang 2pleuropneumoniae; Escherichia coli, Yersinia pestis and
Pseudomonas fluorescens) [3,4] DspB belongs to
fam-ily 20 of the glycoside hydrolase classification scheme,
members of which are exo-acting hexosaminidases that
cleave terminal monosaccharide residues from the
non-reducing end The substrate of DspB is a
hexosamine-containing matrix polysaccharide consisting of a linear
homoglycan of N-acetyl-d-glucosamine residues in
b(1,6)-linkages (PGA) [5–9] DspB catalytic activity
has been investigated using PGA from E coli, which
showed that the reaction products included GlcNAc
and other unassigned GlcNAc oligomers [4] In an
ear-lier study, we reported the crystal structure of DspB in
complex with glycerol and an acetate at the active site,
which suggested that the active-site architecture of
DspB is similar to that of the family 20
b-hexosami-nidases [10] However, very little information is
avail-able on the mechanistic aspects of DspB
It has been proposed that family 20 and the closely
related family 18 b-hexosaminidases follow a retaining
mechanism (Fig 1) with anchimeric assistance
pro-vided by the C2-acetamido group of the substrate and
an acidic active-site residue acting as the catalytic acid
[11,12] A unique feature of this mechanism is the
acet-amido group acting as the nucleophile in catalysis
Interestingly, a substrate-assisted mechanism is
opera-tive in other sequentially dissimilar but functionally
similar enzymes such as family 56
hyaluronid-ase (EC 3.2.1.35) and family 84 O-GlcNAcase
(EC 3.2.1.52) [13,14] Using the crystal structure of
DspB enzyme [10], we identified residues at subsite)1
of the active site that are likely to be involved in
catal-ysis and⁄ or substrate binding We generated a
struc-ture-based sequence alignment through superposition
of the active-site regions of the reported crystal
struc-tures of b-hexosaminidases [15–18] and noted that, in
addition to the well-conserved pair of acidic residues
D–E at positions 183 and 184 (DspB numbering),
acidic residue E332 is also structurally conserved
(Fig 2A,B) Juxtaposition of this residue appears to be
suitable for exerting a significant role in substrate
binding In b-hexosaminidases, highly conserved tryp-tophan residues at subsite)1 create a binding pocket for the terminal GlcNAc, especially the N-acetyl group Because there is a clear difference in the sub-strate specificity between DspB [acts on b(1,6)-linked poly(N-acetylglucosamine)] and the other members of the family 20 b-hexosaminidases [act on b(1,4)-linked N-acetylglucosamine], we tested the roles of conserved and nonconserved residues at the active site using site-directed mutagenesis Our results demonstrate that: (a) DspB follows a substrate-assisted mechanism proposed for the family 20 b-hexosaminidases with participation
of the conserved acidic pair D183 and E184; (b) the aromatic residues W237 and Y278 along with D183, play a role in hydrolysis and assist the N-acetyl group
to properly orient itself to take part in catalysis; (c) the aromatic residue Y187, which is part of a loop at subsite +1, might play a role in the specificity exhib-ited by DspB; and (d) three residues, R27, E332 and W330 interacting with the GlcNAc at the C4 side pro-vide transition state stabilization during hydrolysis
Results
Mutations were performed using the primers listed in Table 1 Wild-type DspB and the mutants were gen-erated with a His6 tag to facilitate purification using metal-affinity chromatography as described previously [10] SDS⁄ PAGE analysis of the cell lysates indicated that all the mutant enzymes were present in a solu-ble form and were expressed at levels comparasolu-ble with wild-type DspB All proteins were purified to homogeneity using Ni2+-affinity chromatography with yields ranging from 20 to 30 mgÆL)1 of culture Our earlier studies had shown that the half-life of DspB was 3–4 h at 37 C [19] However, DspB was stable and retained its activity for several months when stored in buffer comprising 50 mm sodium phosphate, pH 5.8, 50 mm NaCl and 50% glycerol at )20 C Purified enzymes were stored in this buffer until further use
Fig 1 Hydrolysis mechanism proposed for family 20 b-hexosaminidases In this substrate-assisted mechanism one acidic residue, Glu184, acts as the acid ⁄ base The nucleophile is the N-acetyl group of the substrate and is assisted by Asp183.
Trang 3Role of active-site acidic residues
Comparison of the amino acid sequence and crystal
structure of DspB with other family 20
b-hexosaminid-ases clearly suggests that three acidic residues, D183,
E184 and E332, are highly conserved (Fig 2B)
Among these, the residues equivalent to D183 and
E184 in other family 20 b-hexosaminidases have been
shown to play a critical role in the catalytic reaction
[11,12,17] Thus, the E184 equivalent has been
proposed to act as the acid⁄ base catalyst, whereas the
D183 equivalent has been suggested to orient the
N-acetyl group [11] In general, in family 20
b-hexos-aminidases, the E184 equivalent is juxtaposed close to
the anomeric center, whereas the D183 equivalent is
well positioned to interact with the N- of the
acetam-ido group Many crystal structures also show that a
glutamate residue (equivalent to E332) is located in
close proximity to the C3- and C4-hydroxyl groups on
the opposite side of the bound sugar at subsite)1 To
investigate the proposed role of the D183 and E184
acidic residues and the potential role of E332 in DspB,
these residues were replaced by either N or Q and their
biochemical properties analyzed using the substrate
p-nitrophenyl-b-d-N-acetylglucosamine (pNPGlcNAc)
All three acidic mutants display significantly reduced
specific activities as shown in Table 2 The specific
activity of the catalytic residue mutant E184Q was
122-fold less, and that of D183N was 11 000-fold
lower than wild-type The observed results for the
mutant E184Q are entirely consistent with loss of the
acid–base residue, whereas the significant loss in
activ-ity exhibited by D183N is consistent with its proposed
role as a residue that activates⁄ orients the N-acetyl group to act as a nucleophile [11] Inhibition studies were carried out with the mechanism-based inhibitor NAG-thiazoline [17] IC50 for inhibition was found to
be 147 ± 5 lm compared with 47 ± 4 lm for the jack bean enzyme Inhibition of DspB by NAG-thiazoline
is consistent with an anchimeric assistance mechanism occurring during catalysis in DspB because NAG-thiazoline is a mechanism-based inhibitor of b-hexos-aminidases The 2700-fold lower specific activity
Glu332 Glu184
Asp183
A
B
Fig 2 Sequence and structure alignment of b-hexosaminidases
with DspB (A) Multiple amino acid sequence alignment of DspB
and b-hexosaminidases whose crystal structures have been
reported Subsite )1 residues are indicated by an asterisk (*) Note
the conserved residues WXE near the C-terminus corresponding to
W330 and E332 Although all four aromatic residues, W216, W237,
Y278 and W330 are conserved, the sequence homology in the
region around Y187 of DspB is weak This region might correspond
to residues in subsite +1 and contribute to the substrate specificity
being b(1,6) in DspB Sequences shown are: DspB, 1yht [10];
chito-biase from Serratia marcescens 1qbb [20]; b-hexosaminidase from
Streptomyces plicatus, 1hp5 [17]; human lysosomal
b-hexosamini-dase isoform b, 1now [16]; human b-hexosaminib-hexosamini-dase from placenta,
b-chain, 2gjx [30] The secondary structure as observed in DspB is
shown on top Arrows represent b strands and cylinders represent
helices (B) Superposition of the various crystal structures of
b-hexosaminidases using the suite SWISS - PDBVIEWER [31] The fit of
D183, E184 and E332 residues in the proximity of the subsite )1 is
shown Structures shown are: DspB; PDB Code: 1yht (gray) [10];
PDB Code: 2gjx (red) [30]; PDB Code: 1hp5 (green) [17]; PDB Code:
1now (blue) [16]; PDB Code: 1qbb (yellow) [20].
Trang 4exhibited by E332Q suggests that this residue plays a
significant role in catalysis The location of this
resi-due, although away from the anomeric carbon, might
be critical in providing necessary stabilization in the
transition state while the terminal GlcNAc is
undergo-ing conformational changes durundergo-ing catalysis (Fig 2B)
To further substantiate our observations, kinetic
parameters were determined for the mutants using
pNPGlcNAc as substrate (Table 2) All three mutants exhibited substantially lower Km and significantly reduced kcat values resulting in lower catalytic effi-ciency (kcat⁄ Km) for the mutant enzymes (71-fold lower for E184Q, 13 333-fold lower for D183N and 2000-fold lower for E332Q) compared with wild-type All three mutants required higher enzyme concentrations
to exhibit measurable enzyme activity compared with
Table 1 Primers used for mutational analysis of the active-site residues.
Table 2 Specific activities and kinetic parameters of DspB and its variants for hydrolysis with pNPGlcNAc Kinetic assays were performed with pNPGlcNAc as described in Experimental procedures Reactions were carried out in NaCl ⁄ P i at 37 C The molar absorptivity of p-nitro-phenol used 9900 ( M )1Æcm)1) at 405 nm Results are the average of three independent experiments Standard errors for the values of
appar-ent K m and k cat are given in parentheses ND, not determined because the reaction was too slow to be detected.
Enzyme
Conc (l M ) Specific activity
k cat
(min)1)
K M
(m M )
k cat ⁄ K M
(min)1Æm M )1)
Wild-type
Y187A
Y278A
a No measurable activity for the substrate MuGlcNAc.
Trang 5wild-type (E184Q, 1 lm; E332Q, 12 lm; and D183N,
25 lm compared with 0.1 lm for wild-type; Table 2)
In addition, we also used another substrate,
4-methyl-umbelliferyl-b-d-N-acetylglucosamine (MuGlcNAc), to
study the effect of leaving aglycone (Table 2)
Interest-ingly, this substrate was also poorly hydrolyzed by the
wild-type enzyme and required a higher enzyme
con-centration (4 lm), compared with 0.1 lm for
pNPGlc-NAc There was no measurable activity for the mutant
enzymes, D183N, E184Q and E332Q
The pH activity profiles of the wild-type and variant
enzymes were also determined using pNPGlcNAc and
are shown in Fig 3 The wild-type enzyme exhibited
an approximately bell-shaped profile with a pH
opti-mum of 5.8, which might be interpreted as resulting
from the ionization of two residues with pKavalues on
either side of the maximum The apparent pKa value
for the basic limb corresponding to the acid⁄ base of
the wild-type profile was estimated to be 6.8 ± 0.1
Interestingly, the pH optimum for the E184Q variant
is shifted to 6.8 with an apparent pKa of 8.3 ± 0.1 for
the basic limb consistent with the substitution of E184
carboxyl group to an amide The pH activity profile of
E184Q mutant supports its presumed role in catalysis
as the acid⁄ base (Fig 1) The extremely low activity of
D183N and E332Q variants for the pNPGlcNAc
substrate prevented us from obtaining a pH profile for
these mutants
Role of aromatic residues at the active site One of the characteristics of family 20 b-hexosaminid-ases is the substrate-binding pocket provided by a number of conserved tryptophan residues As shown
in Fig 2A, W216, W237 and W330 are well con-served and together create a hydrophobic pocket into which the N-acetyl group of the GlcNAc moiety at subsite)1 binds [15] Together with the acidic resi-dues, such binding juxtaposes the acetamido group for neighboring-group participation and enables intra-molecular nucleophilic attack at the anomeric center
of the sugar molecule at subsite )1 [16] Residue Y278 occupies a space close to the bound acetyl group and in several crystal structures, including DspB, is known to interact with the O of the acetyl group via a hydrogen bond Residue Y187 is unique
to DspB in that it is located in a mobile loop that is disordered in the crystal structure (PDB code 1YHT) [10] and appears to be present at the entrance to the catalytic site To investigate the roles of these aro-matic residues in DspB, we designed mutations at these positions (Table 1) and the results of the bio-chemical analysis are given in Table 2 Clearly, muta-tion of the tryptophan residues at posimuta-tions 237 (W237A) and 330 (W330Y), which create the N-ace-tyl-binding pocket, had a drastic effect on enzyme activity We observed that even at concentrations of 9–16 lm, enzyme activity was undetectable using pNPGlcNAc as the substrate (Table 2) Both mutants, W237A and W330Y, were also ineffective when MuGlcNAc was used as a substrate
Mutation of the two tyrosines at the active site (Y187 and Y278) showed lower specific activity than the wild-type (Table 2) by as much as 33-fold (Y187A)
or 176-fold (Y278A) Mutant Y187A has a higher Km
and a lower kcat in the hydrolysis of pNPGlcNAc (Table 2) The lower kcat⁄ Km value (40-fold less for pNPGlcNAc and 4.9-fold less MuGlcNAc) for Y187A suggests that the mutation at Y187 affects substrate binding Involvement of this residue, which is farther away from subsite)1 (6 A˚), suggests that DspB might have multiple subsites for substrate binding Mutant Y278A, by contrast, showed a greater decrease in
kcat⁄ Km value (923-fold for pNPGlcNAc and 45-fold for MuGlcNAc) compared with wild-type suggesting that the hydrogen bond to the acetamido group is sig-nificant for enzyme activity Because this hydrogen bond involves the hydroxyl group of Y278 and car-bonyl oxygen of the acetamido group, the reduction in the kinetic parameters suggests that Y278 might partic-ipate in orienting the acetyl group in conjunction with the D183 residue
0
50
100
150
200
250
300
wild type
E184Q
R27K
pH
0 3 6 9 12
15
R27K E184Q
pH
Fig 3 pH dependence of wild-type DspB and mutants The pH
profile was measured using pNPGlcNAc as the substrate at pH
val-ues of 3.5–10.0 The solid lines refer to the fitted curve through the
data points using a Gaussian form using GraphPad PRISM 3 (Inset)
Bell-shaped curve for E184Q showing the shift in optimum pH for
the mutant Activity values were arbitrarily scaled by a factor of 10
to show the Gaussian fit of the profile The pH profile for the
mutants of D183N and E332Q could not be measured due to their
very low activities Note that no pH shift was observed for the
R27K mutant.
Trang 6Role of R27 at the active site
b-Hexosaminidases have one arginine at the active site,
equivalent to R27 of DspB, that is structurally
con-served and involved in substrate binding [11,12]
Crystal structure analyses of hexosaminidases with
substrate⁄ inhibitor complexes have shown that
R27-equivalent arginine enters into a hydrogen-bonding
interaction with C3 and C4 oxygen atoms In the
crys-tal structure of DspB with glycerol at the active site, it
was observed that R27 hydrogen bonds with the
hydroxyl groups of glycerol [10] As shown in Table 2,
a conservative mutation at position 27 of DspB
(R27K) affects the activity less than a nonconservative
mutation (R27A) Nevertheless, both substitutions
ren-dered the enzyme significantly less active with a
2400-fold reduction in kcat⁄ Km for the R27K mutant
(5.5 lm) and a 1714-fold reduction for R27A (6.0 lm)
Both mutants were ineffective against MuGlcNAc
Biofilm detachment and hydrolytic activities
of DspB and its mutants
We previously used the biofilm detachment assay in
which the substrate is the PGA polymer to study the
activity of DspB on biofilms of various bacteria [1,2,19] The PGA polymer from S epidermidis is a natural substrate with a chain length of 130 GlcNAc residues in b(1,6)-linkages [5] We used this biofilm-detachment assay to validate the hydrolytic activities
of the active-site mutants using biofilm from S epide-rmidis As shown in Fig 4A, the E184Q mutant showed very low activity that was not measurable even
at an enzyme concentration of 1.4 lm Although both D183N and E332Q variants are less effective than E184Q in the hydrolysis of pNPGlcNAc, these variants behaved differently towards the detachment of S epi-dermidis biofilm Both D183N and E332Q mutants exhibited biofilm-detachment activity [time required for 50% detachment, T50, is 7.2 min for E332Q at 0.055 lm (163-fold lower) and 8.0 min for D183N at 0.275 lm (28-fold lower than wild-type; Table 3)] Mutants W237A and W330Y exhibited biofilm-detach-ment activity with a T50 of 1 min for W330Y and
9 min for W237A, each at an enzyme concentration
of 0.275 lm (Fig 4B) In contrast to pNPGlcNAc hydrolysis, the biofilm-detachment activity of the tyro-sine mutants is not severely affected (Fig 4C) Thus, the T50 for the mutant Y187A is 5.5 min at 0.022 lm and for the Y278A mutant is 2.8 min at 0.055 lm
0
1
2
3
4
5
E332Q (0.055 µM) E184Q (1.375 µM) wild type (0.011 µM)
D183N (0.275 µM)
Absorbanceat 590 nm Absorbanceat 590 nm
0 1 2 3 4 5
W237A (0.275 µM) Wild type (0.011 µM) W330Y (0.275 µM)
0 1 2 3 4
5
R27A (0.056 µM) R27K (0.055 µM) Wild type (0.011 µM)
0
1
2
3
4
5
Y278A (0.055 µM) Y187A (0.022 µM) wild type (0.011 µM)
Time (minutes)
Time (minutes)
Time (minutes)
Time (minutes)
DspB Y187A W237A Y278A W330Y D183N E184Q E332Q R27A R27K
0 10 20
0 25 50 75
100 GlcNAc
Enzyme
Fig 4 S epidermidis biofilm detachment activity of DspB and its mutants (A) Biofilm detachment activity of DspB and its acidic variants D183N, E184Q and E332Q Biofilms were treated with NaCl ⁄ P i or the enzyme variant for the indicated time intervals and assayed in 96-well microtiter plates [1,2,19] Biofilms were then rinsed and stained with crystal violet The amount of bound crystal violet dye which is propor-tional to the biofilm mass was quantitated by measuring its absorbance at 590 nm (B) Biofilm detachment activity of the aromatic residue mutants W237A and W330Y (C) Biofilm detachment activity of the aromatic residue mutants Y187A and Y278A (D) Biofilm detachment activity of the mutants R27A and R27K Values plotted correspond to the mean absorbance of triplicate wells Error bars indicate standard deviations calculated using the program PRISM 3 (GraphPad) Legends include the final concentrations of the enzymes used in the detachment assay (E) Plot of relative efficiency of biofilm detachment (shown on the right y-axis) and the amount of GlcNAc generated (left y-axis) by the various mutants in comparison with wild-type DspB.
Trang 7Interestingly, the two mutants R27A and R27K
showed biofilm-detachment activity at concentrations
0.055 lm with T50 5–7 min, which is 20–36-fold less
efficient than the wild-type (Fig 4D) The relative
bio-film-detachment activities of the mutants were
com-pared with the amount of GlcNAc generated due to
hydrolysis of PGA in the biofilm (Fig 4E) One
nota-ble outcome from the GlcNAc-releasing activities of
the R27 mutants is that R27K exhibited much higher
activity than R27A Nevertheless, mutation of residues
that interact with the acetyl group (W237, W330,
D183, Y278) or the catalytic residue E184 showed low
activity in both biofilm detachment and pNPGlNAc
hydrolysis
Discussion
It is generally accepted that family 20 hydrolases such
as chitobiase from Serratia marcescens and human
b-hexosaminidases operate via a retaining mechanism
[17] In such a mechanism, a general acid⁄ base catalyst
plays a dual role by first protonating the departing
aglycone and then deprotonating the incoming water
molecule (Fig 1) Because of a lack of nucleophile
derived from protein, family 20 b-hexosaminidases and
other functionally similar enzymes such family 56
hyaluronidase and family 84 O-GlcNAcase utilize the
C2-acetamido group to help stabilize the incipient
oxazolinium ion at the anomeric carbon [13,14,17,20]
Formation of the cyclic oxazolinium ion intermediate
is assisted by another acidic residue, which interacts
with the nitrogen atom of the N-acetyl group and
helps stabilize the transition state The cyclic
interme-diate formed is then hydrolyzed by the general
base-catalyzed attack of the water at the anomeric center The strongest candidate for the acidic residue that sta-bilizes the transition state and the acid⁄ base residue has been suggested to be a highly conserved DE or
DD pair [21], equivalent to D183 and E184 in DspB Because of the involvement of this pair, family 20, 56 and 84 enzymes have been suggested to follow a sub-strate-assisted mechanism [11,13–16,20] Based on superposition of the available crystal structures of vari-ous b-hexosaminidases with that of DspB structure (PDB code 1YHT), subsite)1 in DspB has been deduced to be formed by residues R27, D183, E184, W216, W237, Y278, W330 and E332 (Fig 5A) Thus, the active-site architecture in DspB, at least at subsite)1, has several salient features that are charac-teristic of b-hexosaminidases exhibiting the substrate-assisted mechanism
Role of acidic residues Among the three acidic residues studied here, D183 is the putative residue that assists the N-acetyl group in neighboring-group participation Replacement of Asp with Asn at position 183 in DspB rendered this mutant inactive Mutations of D183-equivalent residues pro-duced very low enzyme activity in human b-hexosamin-idase (Hex A), Streptomyces plicatus b-hexosaminb-hexosamin-idase and human O-GlcNAcase enzymes Thus, the mutant D354N (human enzyme) exhibited a kcat value that was only 0.04% that of the wild-type [12], whereas D313N (SpHex enzyme) produced a 560 000-fold reduction in the kcat⁄ Km value [11] and that of the O-GlcNAcase exhibited a 6727-fold reduction in
kcat⁄ Km [21] The very low activity of the D313N mutant in SpHex was rationalized by the increased bulk (COO– to CONH2) due to the mutation and⁄ or
by the adaptation of alternate conformations of the N-acetyl group of the terminal GlcNAc residue [11] In addition to the assistance provided to activate the N-acetyl group, it has been suggested that this Asp might control the orientation of the C2-acetamido group The inactivity of the D183N mutant suggests that residue D183 in DspB, like its counterparts in other b-hexosaminidases, participates in cleavage of the glycosidic bond and formation of the intermediate
by assisting the acetamido group during catalysis The conclusion that DspB utilizes a substrate-assisted catal-ysis not only relies on comparison with the work of others, but is also supported by the ability of NAG-thiazoline to inhibit DspB
Our results support the role of E184 in DspB as an acid⁄ base catalyst because the E184Q mutant shows significant reduction in kcat⁄ Km(67-fold lower than the
Table 3 Relative biofilm detachment efficiency of DspB and its
mutants with respect to DspB Values are given in time required
for removal of 50% of biofilm Relative efficiency was calculated
as: 100 · (T 50 for DspB ⁄ T 50 for mutant) · (concentration of
DspB ⁄ concentration of mutant).
Enzyme
Concentration
used (l M )
T 50
(min)
% Relative efficiency
a Data could not be fitted because of very low activity.
Trang 8wild-type) and a shift in the apparent pKa for the
acid⁄ base Mutational studies on E314 from S plicatus
also showed a reduction in the kinetic parameters and
a shift in the pH optimum The acid⁄ base role for this
residue is identical to that of the equivalent
gluta-mate⁄ aspartate residues in family 20
b-hexosaminidas-es and family 56 and family 84 enzymb-hexosaminidas-es
Our findings reveal an important role for E332 in
DspB and possibly other hexosaminidases as well As
shown in Fig 2B, E332 of DspB and its equivalent
res-idues (residue Glu739 in the human Hex enzyme and
residue E444 in the S plicatus enzyme) in
fam-ily 20 b-hexosaminidases occupy the same space on the
opposite side of the bound ligand at subsite)1 These
residues are positioned in space to interact with the
hydroxyl group at C4 Kinetic parameters obtained for
the E332Q mutant, reflecting the loss of activity upon
mutation, unequivocally show that a loss of
transition-state stabilization has occurred In this regard, E332
residue might be acting similar to D300, one of the
three catalytic residues in human salivary a-amylase and other a-amylases that belong to the family 13 hy-drolases [22,23] Overall, the acidic residue mutations
in DspB tend to destabilize the transition state and the oxazolinium intermediate (Fig 1) that might be accu-mulated (low Kmvalues), and undergo a slower hydro-lysis (reduced kcat) by a suitably juxtaposed water molecule
Role of aromatic residues Although subsite)1 is highly conserved in family 20 b-hexosaminidases, significant differences in the active site of DspB should be noted For example, the bind-ing pockets created by the aromatic residues for sub-site)1 in DspB and S plicatus enzyme differ in size and shape (Fig 5B) It is possible that the difference
in pocket shape might lead to subtle differences in the binding of the terminal GlcNAc residue and enable the active site of DspB to be flexible Differences
R27
E332
W330
W237
E184
A
B
D183
GlcNAc
Fig 5 Active site of DspB (A) Active-site residues of DspB and their potential interactions with a docked GlcNAc at subsite )1 GlcNAc was docked onto the DspB active site manually using SpHex structure (PDB code: 1hp5) [17] as a reference Superposition of the crystal structures was carried out using
SWISS - MODEL [31] (B) Superposition of the active-site cavity formed by subsite )1 in DspB (blue) and S plicatus enzyme (yellow) Two views 90 to each other are shown in the left and right panels Note the difference
in the cavity size and shape (207 A ˚ 3 for DspB and 222 A˚ 3 for S plicatus enzyme) suggesting that the binding of the terminal GlcNAc residue might be different in the two enzymes Cavity was calculated using the server at http://www.bioinformatics leeds.ac.uk/cgi-bin/pocketfinder/.
Trang 9in the substrate specificity in DspB versus other
b-hexosaminidases might arise because of differences
in the residues that create subsite +1 In particular,
tryptophan residues W685 (human Hex A) and W408
(S plicatus), respectively, interact with the subsite +1
moiety via hydrophobic-stacking interactions [16,17]
In DspB, an equivalent tryptophan residue is absent
In this regard, the juxtaposition of two loops segment
in DspB (residues 185–191 containing Y187) and a
sec-ond loop (residues 238–247 containing Q244) might be
important As a result, there are two pockets in the
active site, formed by the intrusion of Y187, and
unnatural substrates such as pNPGlcNAc may be
ori-ented to bind in these pockets, albeit poorly (Fig 6)
Another commonly used substrate, MuGlcNAc, is also
poorly cleaved by DspB (Table 2) suggesting that
agly-cone might be critical in DspB The first-order rate
constant for this substrate is 39-fold lower than for
pNPGlcNAc No measurable activity was observed for
the mutants D183N, E184Q, E332Q, W330Y, W237A,
R27A and R27K, whereas values for the other two
mutants, Y187A and Y278A are consistent with their
proposed roles In the case of SpHex enzyme, however,
the kinetic parameters for the two substrates,
pNP-GlcNAc and MuGlcNAc, are comparable (kcat:
193 ± 3 s)1 versus 180 ± 7 s)1; Km: 0.049 ± 0.004
mm versus 0.054 ± 0.03 mm; and kcat⁄ Km:
3900 ± 400 s)1Æmm)1 versus 3300 ± 300 s)1Æmm)1)
[24] The importance of the aglycone in DspB is in contrast to the existing body of work on other fam-ily 20 enzymes that cleave b(1,4)-linked substrates, wherein the aglycone does not appear to have a signifi-cant impact Analysis of the reaction mixtures by TLC with chitin oligosaccharides using DspB and the mutants did not generate any hydrolytic products (data not shown) further substantiating the substrate specificity of DspB The active site of DspB is clearly designed to bind 1,6-linked substrates whereas 1,4-linked substrates and other substrate analogs mimick-ing 1,4-linked substrates either do not bind at all, or bind only poorly
Note that the acetyl group and W237 are involved
in a stacking interaction as has been observed in many b-hexosaminidase crystal structures bound to a ligand Even in DspB, a similar stacking is present between W237 and the bound acetate mimicking the N-acetyl group [10] In the W237A mutant, binding of the ace-tyl group might have been affected thus reducing the enzyme activity against pNPGlcNAc substrate By con-trast, the observed activity of the tryptophan mutants W237A and W330Y in biofilm detachment could be explained because the active site is designed to bind the 1,6-linked substrate as present in the biofilm matrix These results have been further confirmed by total glucosamine assay measuring the hydrolysis rather than the biofilm detachment (Fig 4E) The rela-tive hydrolytic activity, as measured by the release of GlcNAc (Fig 4E), might be considered to reflect true activities of these mutations than the use of pNPGlc-NAc as a substrate It is likely that DspB possesses multiple subsites for GlcNAc residues to bind and pro-vides potential interactions between GlcNAc residues
at subsites +1 and⁄ or +2 and protein atoms
The role of arginine at the active site The available crystal structure data show that R27 is directly involved in substrate binding and helps to dock a GlcNAc at subsite)1 This is facilitated by bridging interactions with the C3- and C4-hydroxyl groups, as observed in the crystal structures of several b-hexosaminidases reported in the literature The lower
kcat value for mutant R27K suggests that R27 might
be involved in stabilization of the transition state [18,25] Interestingly, distinct differences in GlcNAc-releasing activity from the biofilm have been noted for the two R27 mutants (Fig 4E) The low activities of the two mutants with respect to pNPGlcNAc hydroly-sis, compared with the wild-type, may be because this substrate does not truly mimic the natural substrate The difference between substitution with a neutral side
Fig 6 Representation of the surface cavity in DspB The large
substrate-binding pocket is partitioned by the residues Y187 and
Q244 positioned in the middle The residue W237 provides a wall
for a stacking interaction with the bound acetate ⁄ N-acetyl group.
The pocket extends on the right and might be utilized to bind
b(1,6)-linked GlcNAc polymer to generate multiple subsites.
Trang 10chain (R27A) and a charged side chain (R27K)
becomes apparent when one compares
biofilm-detach-ment activities The small increase in efficiency for
mutant R27K (Fig 4D and Table 3) is likely to arise
because of the residual potential stabilization of the
transition state due to retention of the positive charge
(R27 versus K27) However, the biofilm is a complex
environment and detachment activity requires action
on the surface-attached PGA or PGA very close to the
abiotic surface By contrast, when one compares the
GlcNAc-releasing activity of the mutant and wild-type
enzymes, charge retention at the transition state in
R27K clearly reflects the role of R27 in DspB Thus,
R27K exhibits a significantly higher activity (Fig 4E)
than R27A Unlike biofilm detachment, GlcNAc
release can occur from the exposed chains of PGA in
the biofilm each of which consists up to 130
b(1,6)-linked GlcNAc moieties [5] further supporting the idea
that GlcNAc-releasing activity might truly reflect the
roles of these residues in DspB
Active-site residues of DspB and their role
in the substrate-assisted mechanism
The hydrolysis of pNPGlcNAc and the
biofilm-detachment activity exhibited by the mutants studied
clearly suggest that several residues at subsite)1 play
a significant role in the hydrolytic reaction It is well
established that the position of the N-acetyl group is
critical for b-hexosaminidase enzymes to exhibit a
substrate-assisted mechanism Whereas earlier reports
focused on the individual role played by D183 in this
regard, our results show that, in addition to D183,
residues such as W237 and Y278 assist in orienting
the acetyl group by participating in a critical-stacking
or hydrogen-bond interaction with it Mutation of
R27 or E332 has a drastic effect on enzyme activity
Because these residues are on the same side of the
bound ligand and interact with C4 oxygen, the
stabil-ization provided by the residues in the transition state
appears to be critical as the saccharide is undergoing
a conformational change A detailed study on the
probable conformational changes during the
sub-strate-assisted mechanism has suggested that the
ter-minal saccharide changes from a skew conformation
in the Michaelis complex, to an envelope (or perhaps
half chair) conformation in the transition state, and
then to a chair conformation in the intermediate [14]
In this regard, residue W330, also on the side of C4,
provides a wall for the GlcNAc-binding pocket and
thus might work in concert with R27 and E332 to
provide the necessary stabilization in the transition
state Our results suggest that hydrolysis in DspB
occurs via a substrate-assisted mechanism Further-more, the notable differences in the activity of DspB mutant Y187A towards pNPGlcNAc and PGA biofilm from S epidermidis, suggest that DspB might utilize the loop containing Y187 for its substrate specificity In conclusion, our mutational analysis has shown that every residue at the subsite )1 in DspB is essential for optimal activity
Experimental procedures
Expression and purification of DspB and mutants
DspB and its site-specific mutants were expressed and puri-fied using plasmid pRC3 carrying the dspB gene (encoding amino acids 21–381 fused directly to a hexahistidine metal-binding C-terminal tail located downstream from an iso-propyl thio-b-d-galactoside-inducible tac promoter) [10] Mutants of DspB were generated using the primers listed in Table 1 Native and the mutant enzymes were expressed as previously described [10] Briefly, a 2 L Erlenmeyer flask containing 500 mL Luria–Bertani broth supplemented with
30 lg kanamycin per mL was inoculated with 5 mL of an overnight culture of E coli strain Rosetta (DE3) (Nov-agen, Madison, WI) [26] transformed with the plasmid pRC3 containing the appropriate mutation The flask was incubated at 37C with agitation (200 r.p.m.) until
A600¼ 0.6 (c 3 h) Isopropyl thio-b-d-galactoside was added to a final concentration of 0.1 mm, and the flask was incubated for 5 h with agitation Cells were harvested by centrifugation for 15 min at 6000 g
The cell pellet was resuspended in 20 mL lysis buffer (20 mm Tris⁄ HCl, pH 8.0, 500 mm NaCl, 1 mm phenyl-methylsulfonyl fluoride and 0.1% Nonidet P-40) The cell suspension was then sonicated on ice for 30 s (· 5 with
2 min intervals) at 30% capacity with a 30% duty cycle using a Branson model 450 sonicator equipped with a microprobe The cell debris was pelleted by centrifugation (15 000 g for 20 min; 4C), and the supernatant was loaded onto a 3 mL (bed volume) activated Ni–NTA aga-rose affinity column (Qiagen, Valencia, CA) according to the manufacturer’s instructions The column was washed with 50 mL wash buffer (20 mm Tris, pH 8.0, 500 mm NaCl) containing 5 mm imidazole Bound proteins were eluted with 25 mL wash buffer containing 50 mm imidaz-ole Fractions of the eluate (5 mL) were collected and assayed for the presence of DspB by SDS–PAGE and Coomassie Brilliant Blue R250 staining [27] Fractions containing pure protein were pooled and dialyzed overnight against water (2· 4 L changes at 4 C) by using a 12–14 kDa cut-off dialysis membrane (Spectra⁄ Por2, Spectrum Laboratories, Inc., Rancho Dominguez, CA), and lyophil-lized The purity and molecular mass of DspB proteins were determined by MS analysis