The position of Glu36 in CsnN174 is equivalent to general base residue in GH19 chitinases, whereas Thr45 is located similarly to the catalytic residue Asp52 of GH22 lysozyme.. Site-direc
Trang 1Variations on a common theme in the lysozyme superfamily
Marie-E`ve Lacombe-Harvey1, Tamo Fukamizo2, Julie Gagnon1, Mariana G Ghinet1,
Nicole Dennhart3, Thomas Letzel3and Ryszard Brzezinski1
1 De´partement de Biologie, Centre d’E´tude et de Valorisation de la Diversite´ Microbienne, Universite´ de Sherbrooke, Canada
2 Department of Advanced Bioscience, Kinki University, Nara, Japan
3 Department for Basic Life Sciences, Technische Universita¨t Mu¨nchen, Freising-Weihenstephan, Germany
The chitosanase from Streptomyces sp N174
(CsnN174) catalyzes the hydrolysis of b-1,4-glycosidic
links in chitosan, a water-soluble derivative of chitin
composed of d-glucosamine (GlcN) with a variable but
minor proportion of N-acetyl-d-glucosamine (GlcNAc)
[1] Research on the enzymatic hydrolysis of chitosan
is driven by the fact that this polymer has numerous
potential applications and that its properties often
depend on its molecular mass [2] CsnN174 belongs to family 46 of the glycoside hydrolases (GH46), endohy-drolase-type enzymes acting via an inverting mecha-nism [3,4] GH46 enzymes belong to the GH-I clan [5] together with lysozymes from family GH24 (the most studied being the lysozyme from T4 phage) Enzymes from these two families share the same catalytic mech-anism and are folded similarly, with two globular
Keywords
chitinase; chitosanase; glycoside hydrolase;
inverting mechanism; lysozyme
Correspondence
R Brzezinski, De´partement de Biologie,
Universite´ de Sherbrooke, 2500 boul de
l’Universite´, Sherbrooke, QC J1K 2R1,
Canada
Fax: +1 819 821 8049
Tel: +1 819 821 8000; ext 61077
E-mail: ryszard.brzezinski@usherbrooke.ca
(Received 22 September 2008, revised 26
November 2008, accepted 3 December
2008)
doi:10.1111/j.1742-4658.2008.06830.x
The chitosanase from Streptomyces sp N174 (CsnN174) is an inverting glycoside hydrolase belonging to family 46 Previous studies identified Asp40 as the general base residue Mutation of Asp40 into glycine revealed
an unexpectedly high residual activity D40G mutation did not affect the stereochemical mechanism of catalysis or the mode of interaction with sub-strate To explain the D40G residual activity, putative accessory catalytic residues were examined Mutation of Glu36 was highly deleterious in a D40G background Possibly, the D40G mutation reconfigured the catalytic center in a way that allowed Glu36 to be positioned favorably to perform catalysis Thr45 was also found to be essential Thr45 is thought to orientate the nucleophilic water molecule in a position to attack the glycosidic link The finding that expression of heterologous CsnN174 in Escherichia coli protects cells against the antimicrobial effect of chitosan, allowed the selec-tion of active chitosanase variants after saturaselec-tion mutagenesis Thr45 could
be replaced only by serine, indicating the importance of the hydroxyl group The newly identified accessory catalytic residues, Glu36 and Thr45 are located on a three-strand b sheet highly conserved in GH19, 22, 23, 24 and
46, all members of the ‘lysozyme superfamily’ Structural comparisons reveal that each family has its catalytic residues located among a small num-ber of critical positions in this b sheet The position of Glu36 in CsnN174 is equivalent to general base residue in GH19 chitinases, whereas Thr45 is located similarly to the catalytic residue Asp52 of GH22 lysozyme These examples reinforce the evolutionary link among these five GH families
Abbreviations
(GlcN) n , b-D-glucosamine oligosaccharide with n monomer units; CsnN174, chitosanase from Streptomyces sp N174; GH, glycoside hydrolase family; GlcN, D -glucosamine; GlcNAc, N-acetylglucosamine.
Trang 2(mostly a-helical) domains separated by a substrate
binding cleft [6–9]
Site-directed mutagenesis studies of CsnN174,
com-bined with crystallography data, catalytic functions to
be assigned to residues Glu22 (the general acid) and
Asp40 (the general base) [10] These residues are
strictly conserved among all GH46 proteins for which
chitosanase activity has been confirmed by biochemical
studies Glu22 is close to the C-terminus of an a helix,
belonging to a central structural core consisting of two
a helices and a b sheet [8] This structure is shared
with GH24 enzymes, and is also found in GH19
chitinases and GH22 or GH23 lysozymes, represented
respectively by the extensively studied chitinase from
barley seeds and lysozymes from hen and goose
egg-white This group of enzymes is sometimes designated
as the lysozyme superfamily [6,8] Despite their
struc-tural similarity and a highly equivalent positioning of
the general acid residue, GH22 enzymes differ from
the others in this group in that they act by a
mecha-nism with anomeric retention [11] Asp40 of CsnN174
is found inside another element of the central
con-served core: a sheet formed by three antiparallel
b strands separated by loops of varying lengths [7]
In contrast to the general acid residue, the general
base residues are not localized in equivalent positions
in these five families [8] In the extreme case, no
resi-due with general base function has so far been
pro-posed for goose egg-white lysozyme [12] Details of the
catalytic mechanism should vary among these
structur-ally related enzyme families
This study was initiated by the observation that a
CsnN174 mutant in which the general base residue has
been substituted by a glycine (D40G) retained a
signifi-cant proportion of the wild-type activity Studies of
residues that caused complete loss of activity in this
mutant led to the identification of two residues with
accessory catalytic functions We developed a method
for revertant chitosanase identification among a
popu-lation of inactive enzyme-encoding genes based on the
discovery that the heterologous CsnN174 expression
protects Escherichia coli against the antimicrobial
effect of chitosan Finally, we discuss the evolutionary
implications of the presence of such accessory catalytic
residues in GH46 chitosanases
Results
N174 chitosanase devoid of Asp40 retains
significant enzymatic activity
In a previous study, we identified Asp40 as the best
candidate for the general base in the inverting
mecha-nism This was supported by its positioning in the 3D structure [7] and the substantial loss of activity observed for enzymes mutated in this position, because the replacement of Asp40 by conservative Glu or Asn residues resulted in a decrease of kcat to 1⁄ 125 and
1⁄ 485 of wild-type, respectively [10] These values were typical of similar mutations in other inverting glyco-side hydrolases [13]
A mutation path was suggested by Brameld and Goddard to rebuild the GH19 barley inverting chitin-ase into a retaining enzyme [14] This set of mutations, resulting from molecular dynamics simulations, included mutation of the general base residue Glu89 into glycine followed by mutation of Gly113 into glu-tamate Although (to our best knowledge) a GH19 enzyme with a retaining reaction mechanism has not been disclosed in the literature, it was worth trying to introduce analogous mutations in CsnN174, consider-ing the similarity of the structural cores among GH46 and GH19 enzymes [8] We thus mutated Asp40 of chitosanase into Gly [15] In preliminary studies, the D40G mutant revealed significant activity, unexpected for an enzyme devoid of its general base catalytic resi-due We further proceeded with kinetic analysis which revealed that Km remained similar to wild-type (Table 1), whereas kcat was 28 times lower than wild-type but, respectively, 4.5 and 17.5 times higher than that of mutants D40E and D40N studied previ-ously [10] The data suggested that D40G mutant chitosanase was impaired in its catalytic activity although its substrate-binding mode remained essen-tially unchanged
Table 1 Specific activities and kinetic parameters of purified wild-type and mutant CsnN174 All specific activities were determined
at a single chitosan concentration (800 lgÆmL)1) Kinetic parame-ters were calculated using the non linear least-square fitting proce-dure for Michaelis–Menten equation in PRISM software v 5.0 ND, not determined.
Enzyme
Specific activity (unitsÆmg)1protein)
K m (lgÆmL)1)
k cat (min)1)
Trang 3Interaction of the D40G mutant with the substrate
could be investigated in several ways First, the
sub-strate-binding ability was assessed by thermal unfolding
experiments (Fig 1) (GlcN)3 binding to the wild-type
enzyme increased the transition temperature (Tm) by
5.7C, and its binding to D40G increased Tm by
2.8C Thus, (GlcN)3 binding to D40G stabilizes the
protein structure to a similar extent as in the case of
the wild-type enzyme
The mode of hydrolysis of glucosamine
oligosaccha-rides [5,15,16], however, provides insight into the
inter-action of the enzyme with the substrate during the
reaction, because a mutation of a residue involved in
substrate binding is expected to result in an altered
time course of hydrolysis The reaction time course of
D40G mutant enzyme was thus investigated with
(GlcN)5 and (GlcN)4 substrates and monitored by real-time MS [15] As shown in Fig 2, the specific activity of D40G chitosanase, determined from the degradation rate of these substrates was found to be 7.4Æmin)1 (wild-type = 385Æmin)1) for (GlcN)5 and 3.1Æmin)1(wild-type = 140Æmin)1) for (GlcN)4 In both cases, the D40G chitosanase degradation rate is 2% that of wild-type, which is in the range obtained with high-molecular mass chitosan substrate (Table 1) The time course profile of hydrolysis by D40G mutant is similar to that of the wild-type (Fig 2), indicating that this mutant is not impaired in substrate binding A control experiment with (GlcNAc)6 indicated that nei-ther wild-type nor D40G chitosanase are able to cleave GlcNAc–GlcNAc bonds (data not shown)
The stereochemistry of the D40G chitosanase reac-tion was investigated by 1H-NMR As shown in Fig 3, D40G mutant is still an inverter, because the time course of anomer formation is essentially the same as for the wild-type In D40G, the water mole-cule was found to attack the C1 carbon of the transi-tion state sugar residue from the side identical to that
in the wild-type [3]
It was shown recently that a mutation of the gen-eral base residue can be rescued by sodium azide in retaining glycoside hydrolases [17] and also in an inverting a-glycosidase [18] We thus investigated the effect of azide ion on the activity of D40G mutant chitosanase The time courses of (GlcN)6 degradation
in the absence or presence of sodium azide (0.65 and 2.6 m) are shown in Fig 4A The rate of (GlcN)6 degradation was significantly enhanced by the addi-tion of the azide ion The effect of the azide concen-tration on the reaction rate, shown in Fig 4B, clearly demonstrates that the rate enhancement depends upon the azide concentration The results indicate that Asp40 acts as a catalytic base, which activates a water molecule
In summary, substitution of the general base Asp40
by glycine resulted in an enzyme that is distinguished from wild-type only by a lower activity, without changing the mechanism of hydrolysis or the mode of interaction with substrate
Glu36 as a possible alternative general base residue
A possible explanation of the higher activity of D40G chitosanase compared with mutants D40N or D40E was that the mutant D40G reconfigured its three b-strands motif such that another residue could become localized in a favorable position to perform catalysis Glu36 was found to be the best candidate
Fig 1 Thermal unfolding curves of wild-type (A) and D40G (B)
chitosanases in the presence or absence of (GlcN) 3 The enzyme
and the trisaccharide were mixed in 50 m M sodium acetate buffer
pH 5.5 The final concentrations are 2.3 l M for the enzyme and
2.3 m M for the saccharide The unfolding process was monitored
by CD at 222 nm.
Trang 4because its side chain points towards the
substrate-binding cleft (Fig 5A) Glu36 appears to be a minor
player in the wild-type configuration, because its
sub-stitution by Asp, Asn, Gln or even Ala had minor effects on activity, decreasing the catalytic constant at most by one third and slightly increasing the Kmvalue
A B
C D
Fig 2 Time courses of (GlcN) 5 and (GlcN) 4 hydrolysis catalyzed by wild-type and D40G endochitosanases monitored by real-time MS The enzymatic reactions were carried out in 10 m M ammonium acetate-containing aqueous solutions pH 5.2 at 20 C (A) (GlcN) n hydrolysis time courses obtained for wild-type endochitosanase (5.0 n M ) catalyzed reaction performed with 25.0 l M of the substrate (GlcN) 5 (100% = 2.3 · 10 6
counts); (B) (GlcN) n hydrolysis time courses obtained for D40G endochitosanase (200 n M ) catalyzed reaction performed with 25.0 l M of the substrate (GlcN)5(100% = 2.2 · 10 6 counts); (C) (GlcN)nhydrolysis time courses obtained for wild-type endochitosanase (5.0 n M ) catalyzed reaction performed with 25.0 l M of the substrate (GlcN)4(100% = 2.5 · 10 6 counts); (D) (GlcN)nhydrolysis time courses obtained for D40G endochitosanase (200 n M ) catalyzed reaction performed with 25.0 l M of the substrate (GlcN) 4 (100% = 2.8 · 10 6
counts) (A) and (C) were adapted from Dennhart et al [15] with permission.
Fig 3 Anomer production from the D40G mutant chitosanase hydrolysis of (GlcN) 6 (A) Time-dependent 1 H-NMR spectra (B) Time course of anomer production The enzymatic reaction was conducted in 50 m M
deuterated sodium acetate buffer pH 5.0 in
an NMR tube thermostated at 30 C.
Trang 5Fig 4 Chemical rescue experiments (A) Time courses of the enzymatic degradation of (GlcN) 6 by D40G in the absence or pres-ence of sodium azide (0.65 and 2.6 M ) The enzymatic reaction was conducted in 50 m M sodium acetate buffer pH 5.0 and at 40 C The enzyme concentration was 8.5 l M Only some examples of tested concentrations are shown (B) Effect of sodium azide con-centration on the reaction rate of D40G (C) Time courses of the enzymatic degradation of (GlcN) 6 by E36A + D40G in the absence
or presence of sodium azide (2.3 M ).
A
B
Fig 5 (A) Structural view of the active site cleft of chitosanase Csn-N174 The image represents a portion of the chain A from 1CHK file in Protein Data Bank [7] L(1–2); loop between sheets b-1 and b-2; L(2–3), loop between sheets b-2 and b-3 Asp57, Glu197 and Glu201 are residues involved in chitosan substrate binding at )2, )1 and +2 subsite, respectively [37] The model was drawn using PYMOL software (version 0.99; DeLano Scientific, San Fran-cisco, CA, USA) (B) Alignment of portions of the primary structure
of GH46 chitosanases including active site residues Numbering refers to the distance of the first residue from the N-terminus of the mature protein (Csn-N174; chitosanase from B circulans MH-K1) or of the precursor protein as stored in GenBank (other chitosanases) Arrows indicate the residues discussed in this work Symbol explanation (bacterial names followed by accession num-bers for GenBank database): BAC_CIRC, B circulans MH-K1 (D10624); BAC-EHIM, Paenibacillus ehimensis EAG1 (AB008788); BUR_GLAD, Burkholderia gladioli (AB029336); BAC_SUBT, Bacil-lus subtilis (U93875); BAC_AMYL, Bacillus amyloliquefaciens (ABS75305); BAC_KFB, Bacillus sp KFB-CO4 (AF160195); PBCV-1, Chlorella virus 1 of Paramecium bursaria (U42580); CVK2, Chlorella virus CVK2 (D88191); CsnN174, Streptomyces sp N174 (L07779); NOC_N106, Nocardioides sp N106 (L40408); STR_COEL, Strepto-myces coelicolor A3(2) (AL109849.1 ORF SC3A3.02).
0.12
0.1
0.08
0.06
0.04
0.02
0
0.12
0.1
0.08
0.06
0.04
0.02
0
0.12
0.1
0.08
0.06
0.04
0.02
0
Reaction time (min)
160 200 240
0 40 80 120 160 200 240
0 40 80 120 160 200 240
(GIcN)2
(GIcN)6
(GIcN)3
(GIcN)3
(GIcN)6
(GIcN)2 (GIcN)4
(GIcN)2 (GIcN)4
(GIcN)3 (GIcN)6
A
(GIcN)4
B 0.6
0.5
0.4
0.3
0.2
0.1
0
Sodium azide (M)
Reaction time (min)
(GIcN)6
(GIcN)3 (GIcN)4
(GIcN)6
(GIcN)3 (GIcN)4
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
0 100 200 300 400 500 600 700
0 100 200 300 400 500 600 700
Trang 6(Table 1) The effect of the E36 mutation was quite
different in the enzyme with glycine-substituted Asp40
The kcatof the double mutant E36Q + D40G is more
than five times lower than that of the single mutant
D40G and 18 times lower for E36A + D40G,
putt-ing it into the very low range observed for D40N or
D40E, whereas Km in both these double mutants is
increased only by a factor of 2 The rate of (GlcN)6
degradation was also enhanced by sodium azide in
E36A + D40G The enhancement was less intensive
than that in D40G but significant as shown in Fig 4C
In combination with the D40G mutation, these
data suggest that the carboxylate group of Glu36 is in
position to act as a general base in the inverting
mechanism
Thr45 is essential for catalytic activity both in
wild-type chitosanase and mutant D40G
Residues with hydroxyl groups were found in the
microenvironment of general base residues in some
inverting glycoside hydrolases They are thought to
orientate the nucleophilic water molecule in a position
optimal for catalysis Tyr203 of the inverting GH8
xylanase from Pseudoalteromonas haloplanktis [19] or
Ser190 of GH19 Streptomyces griseus chitinase ChiC
(equivalent to Ser120 in barley seed chitinase) [20,21]
are examples of such residues However, no residue
with this function has been proposed in chitosanases
From this point of view, we examined Thr45 as a
pos-sible candidate, a residue highly conserved in GH46
chitosanases (Fig 5B) Thr45 was first mutated into
His or Glu T45H mutation resulted in a complete loss
of activity, whereas T45E mutant had a very low
resid-ual activity but sufficient to perform kinetic analysis
allowing the conclusion that the loss of activity of
T45E can be explained by a severe decrease of kcat
(Table 1) Interestingly, the activity of this mutant
could not be enhanced by sodium azide (data not
shown)
We then verified whether the Thr45 residue is also
essential in the chitosanase reconfigured by the D40G
mutation Two double mutants were examined:
D40G + T45E and D40G + T45D The D40G +
T45E mutant had only 0.03% of wild-type specific
activity when tested on chitosan substrate; a value
sim-ilar to the single T45E mutant The mutant D40G +
T45D was slightly more active (0.1% of wild-type
activity) Again, kinetic analysis of this double mutant
has shown that the loss of activity was explained by a
dramatic decrease in kcat, although Km remained
simi-lar to wild-type This could be confirmed by the
reaction time course of D40G + T45D mutant
investi-gated with (GlcN)6 substrate and monitored by real-time MS As shown in Fig 6, even if the enzyme con-centration was 500-fold higher in the D40G + T45D reaction (Fig 6B), time course profiles were compara-ble between the wild-type and doucompara-ble mutant This also indicates a dramatic decrease in enzymatic activity
in the double mutant
A
B
Fig 6 Time courses of (GlcN)6 hydrolysis catalyzed by wild-type and D40G + T45D chitosanases monitored by real-time mass spec-trometry The enzymatic reactions were carried out in 10 m M
ammonium acetate-containing aqueous solutions pH 5.2 at 20 C (A) (GlcN)nhydrolysis time courses obtained for wild-type endochi-tosanase (5.0 n M ) catalyzed reaction performed with 25.0 l M of the substrate (GlcN)6(100% = 3.1 · 10 6 counts); (B) (GlcN)nhydrolysis time courses obtained for D40G + T45D endochitosanase (2.5 l M ) catalyzed reaction performed with 25.0 l M of the substrate (GlcN) 6 (100% = 3.0 · 10 6 counts).
Trang 7Saturation mutagenesis analysis of residue 45
including selection on chitosan medium
In order to identify any residues that could replace
Thr45 CsnN174, allowing the enzymatic activity to be
kept at levels close to that of the wild-type enzyme, we
studied the reversion of the inactive mutant T45H by
saturation mutagenesis We constructed a library of
several hundred E coli clones with randomly
intro-duced codons at position 45 of the csnN174 gene
harbored by the plasmid pAlter-csn [10]
Chitosan polymer solubilized in growth medium has
antibacterial activity and severely inhibits the growth of
E coli [22,23] The extent of growth inhibition is
dependent on chitosan concentration, average
molecu-lar mass, the pH of the medium and salt composition
We noticed that growth inhibition could be suppressed
by the expression of CsnN174 in E coli JM109 (data
not shown) This led to the development of a method
for chitosanase revertant selection The composition of
the selective medium was optimized using the mutants
V148T and T45H encoding chitosanases having,
res-pectively, 10% and < 0.1% of wild-type activity
(I Boucher & R Brzezinski, unpublished data, and
Table 1) We used chitosan (0.3 gÆL)1) with an average
molecular mass (Mn) reduced to 15 kDa by enzymatic
hydrolysis, which exhibited a severe antimicrobial effect
against E coli [23] although much more soluble in
aque-ous solutions than native chitosan A low concentration
(5 mm) citrate buffer (pH 6.0) was included in the
med-ium to keep the pH slightly acidic and avoid chitosan
precipitation (usually occurring at pH > 6.5) The
chitosan concentration was adjusted to allow growth of
E colistrains expressing wild-type or V148T
chitosan-ase although inhibiting strains expressing the T45H
chitosanase or harboring the empty pAlter-1 vector
During the optimization of the selection medium, we
observed that growth inhibition was highly dependent
on the bacterial density on the Petri plates We further
adjusted the monovalent (Na+) and divalent (Mg2+)
ion concentrations for a density of 500 colony forming
units per plate As the saturation mutagenesis library
contained presumably only a small minority of
chito-sanase-positive revertants, some chitosanase-negative
colonies could still grow on chitosan medium due to a
kind of ‘protective effect’ resulting from higher local
cell density We thus estimated the number of
false-positive colonies recovered on this medium by mixing
various proportions of the V148T
chitosanase-express-ing cells and chitosanase-negative cells and platchitosanase-express-ing
them on media with various salt compositions Both
colonies could be distinguished thanks to
supplemen-tation with
5-bromo-4-chloro-3-indolyl-b-d-galactopyr-anoside and isopropyl thio-b-d-galactoside, because the empty pAlter-1 vector directs b-galactosidase syn-thesis in E coli and false positives appeared as blue colonies The lowest proportion of false positives (1⁄ 250) was obtained after the addition of 200 mm NaCl and 3 mm MgSO4 This salt composition was adopted for the revertant selection experiment
After plating the complete T45H-saturation muta-genesis library ( 450 clones) on the optimized chito-san medium, we obtained 55 colonies of putative chitosanase-positive revertants We sequenced csn genes in 15 randomly chosen clones, revealing Thr resi-dues in nine revertants (three ACC codons, three ACT, two ACG and one ACA) and Ser residues in six revertants (three TCT codons, one AGC, one AGT, one ACT) Codon diversity indicated that the muta-genesis has been performed without bias in nucleotide substitution We concluded that the Thr residue could
be replaced only by Ser, showing the importance of the hydroxyl group in position 45
We purified the T45S chitosanase mutant after its introduction into Streptomyces lividans This mutant was quite active, keeping 71% of specific activity of the wild-type enzyme This confirmed the utility of the chitosan medium for isolation of chitosanase rever-tants Kinetic analysis showed that the T45S mutation results in almost unchanged Km and slightly decreased
kcat(Table 1)
Discussion
Proposed functions for E36 and T45 residues in CsnN174
In this study, we confirmed that Asp40 functions as a general base in CsnN174 catalysis, because the activity
of mutants devoid of this aspartate (D40G and E36A + D40G) can be enhanced by sodium azide The lack of effect of sodium azide on double mutant D40G + T45E activity indicates that the rate enhance-ment observed in the D40G single mutant is derived from complementing the D40 function In this case, the azide ion acts as a general base which enhances the nucleophilicity of the water molecule, as proposed by Miyake et al [18] Unexpectedly, substitution of Asp40
by Gly resulted in an enzyme with a residual activity much higher than predicted for a mutation involving a catalytic residue Asp40 is localized on a loop between the b-1 and b-2 strands of CsnN174 (Fig 5A) Analo-gous loops in related glycosyl hydrolases such as T4 lysozyme or barley chitinase show a conformational diversity, indicating that the loop is potentially mobile [7,8]; a tendency accentuated further by the Asp40 to
Trang 8Gly substitution, because this results in the addition of
a fourth glycine residue to this loop, in addition to
Gly39, Gly41 and Gly43 already present in wild-type
Two of these glycines (Gly41 and Gly43) are present
in most GH46 chitosanases (Fig 5B) suggesting that
the flexibility of this loop is important Finally,
exami-nation of the structure with the what if program [24]
suggested that the Asp40 to Gly mutation eliminates
the salt bridge-type of interaction with the Arg42
resi-due observed in the wild-type enzyme, allowing for
further loop mobility It is then likely that the Glu36
residue, localized in the neighboring b-1 strand
(Fig 5A) could be placed in a position competent for
catalysis In the native chitosanase crystal, the distance
between the catalytic carboxylates (13.8 A˚) is greater
than that usually observed in inverting glycoside
hydrolases, so the binding of substrate must induce a
substantial conformational change to allow catalysis
Glu36 carboxylate is localized 14.9 A˚ from the general
acid residue Glu22, but after the reconfiguration
result-ing from substrate bindresult-ing it could be in position to
perform catalysis To date, the exact conformational
changes occurring after substrate binding could not be
described in GH46 chitosanases, because co-crystals
with substrate could not be obtained; the same
difficul-ties being reported for the structurally related GH19
chitinases [4,7,20,21,25]
Whereas mutations of Asp40 allowed for substantial
residual activity, those of Thr45 had more severe
con-sequences Mutations involving Thr45 reduced the
activity by at least three orders of magnitude and they
had equally severe consequences when introduced in
the enzyme reconfigured by the D40G mutation The
T45 residue appears to be essential for catalysis
Satu-ration mutagenesis revealed however that the residue
can be replaced by a serine with a very moderate loss
of activity, suggesting the importance of a hydroxyl
group but with some tolerance regarding its exact
position Interestingly, although this threonine is
highly conserved among GH46 chitosanases, one
sequenced chitosanase (from Paenibacillus ehimensis;
Fig 5B) has a serine in the corresponding position
[26], which indirectly confirms our revertant analysis
Possibly, the sulfhydryl group in a T45C mutant
could also adequately orientate the water molecule
resulting in decent enzyme activity The absence of
such a mutant among the revertants isolated after
saturation mutagenesis could simply result from
statis-tical probability, but we also remark that residue 45
in CsnN174 is in the immediate proximity of residue
Cys52 Mutation of Thr45 to cysteine could result in
the creation of a disulphide bond with Cys52, making
the sulfhydryl group unavailable for the orientation of
the water molecule and implying loss of enzymatic activity
Thr45 of CsnN174 lies in a position analogous to Thr26 in T4 lysozyme [8], an extensively studied resi-due T26H mutation resulted in conversion of an inverting enzyme into a retaining one [27], whereas T26E mutation resulted in an inactive enzyme forming
a covalent bond with the substrate [28] None of these effects was observed in the corresponding CsnN174 mutants Structural differences between CsnN174 and T4 lysozyme could account for this different behavior, because the mutual positions of the discussed threo-nines and the general base residues (Asp40 in CsnN174 and Asp20 in T4 lysozyme) are not totally equivalent; the hydroxyl group being closer to the general base carboxylate in T4 lysozyme (3.6 A˚) than in CsnN174 (4.9 A˚)
Another explanation for this different behavior was raised by Zechel and Withers [29]: the retaining mecha-nism of the T26H mutant of T4 lysozyme could involve the acetamide group of the substrate, as observed in GH18 or GH20 enzymes hydrolyzing chitin polymers As the mutation effects are, in our case, observed with GlcN oligomers lacking GlcNAc residues, this is unlikely for CsnN174 Besides these differences in mutant behavior, the requirement for a hydroxyl in residue 45 in CsnN174 and the almost complete loss of activity in mutants indicate that posi-tioning of the attacking water is a plausible function for this residue In barley chitinase, Ser120 is thought
to play the same role [21] and the alignment of pri-mary structures of GH19 chitinases (not shown) reveals that this serine is replaced in many enzymes by
a threonine Interaction of these hydroxyl amino acids with water is observed in the crystal structures of the GH46 chitosanase from Bacillus circulans MH-K1 (res-idue Thr60) and the GH19 chitinase of S griseus (Ser190) [4,20]
Catalytic residues in the lysozyme superfamily: variations on a common theme
It is now generally accepted that strict positioning of the catalytic base is not required for inverting glycosid-ases [30] This flexibility results in a variety of confor-mations for the residues supporting the ‘nucleophilic side’ of the catalytic mechanism observed in the lyso-zyme superfamily A closer look at the three b-strands segment of the conserved structural core in this super-family [8] reveals a small number of key structural ele-ments, the residues of which play various functions depending on the enzyme family For example, Glu36
of CsnN174 discussed here lies in a position equivalent
Trang 9to the general base residue of barley chitinase [7,21]
and to the nucleophile of the recently characterized
invertebrate-type lysozyme from Tapes japonica [31]
whereas Thr45 is localized similarly to the nucleophile
of hen egg-white lysozyme [8] Further examples are
shown in Table 2 During their evolution from a
hypo-thetical common ancestor, each group of enzymes
selected the best positions for essential catalytic
resi-dues choosing among a small number of possibilities;
optimizing their configuration to perform hydrolysis
on a particular substrate in a given condition This
‘mosaic’ of positions remains in sharp contrast with
the invariant position occupied by the general acid
residues in the entire superfamily (Table 2)
Chitosanase as a resistance determinant against
antimicrobial action of chitosan
The finding that a heterologous chitosanase can protect
E coli against the antimicrobial activity of chitosan is
novel and raises the possibility of a new function for
chitosanases As described by several authors [2,23,32]
chitosan shows its maximal antimicrobial effect against
E coliat relatively high molecular mass, whereas
chito-san oligosaccharides or short-chain chitochito-san forms
(< 4 kDa) have no inhibitory effect Such a pattern is
also observed for some other bacterial species By
shortening the chain length of chitosan, chitosanase
could function as a resistance factor against the toxic
effect of chitosan Besides a strictly metabolic function,
consisting of the endohydrolysis of high molecular
mass chitosan into oligosaccharides that can be
trans-ported inside the cell to be used as C and N source,
chitosanase could also play the role of a stress
enzyme, protecting the microbial cells against
chito-san This possibility deserves further studies Formal
genetic experiments with chitosanase-producing micro-organisms are in progress in our group
Experimental procedures
Bacterial strains and plasmids
zDM15]) were used for routine plasmid propagation and as hosts in
D40G + T45E, T45E + T45D mutants (Promega, Madi-son, WI, USA) E coli strain DH5a (F) u80lacZDM15
rou-tine plasmid propagation and as host in site-directed muta-genesis procedures of E36A, E36Q and D40G + E36A Recombinant strains of S lividans TK24 were used for chitosanase production [10] The vector pAlter-1 (for site-directed mutagenesis of D40 and T45 mutants), the vector pUC19 (for site-directed mutagenesis of residue E36) and the shuttle vector pFD666 have been described previously [10,33,34] In some experiments, pFD ES, a smaller deriva-tive of pFD666, kindly provided by E Sanssouci and
C Beaulieu, was used as vector for expression of mutated chitosanase genes This derivative has been obtained by pFD666 digestion with AclI and NruI followed by intramo-lecular ligation
Site-directed mutagenesis
The procedure used to generate mutants D40G, T45E,
described previously [10] A variant of this procedure has been used to perform saturation mutagenesis of the T45
Table 2 Key structural motifs for active site residues in the lysozyme superfamily GH 19, 23, 24, 46 enzymes act by inverting mechanism; GH22 enzymes act by retaining mechanism.
Structural motif a CsnN174 (GH46) T4 lysozyme (GH24)
Hen egg-white lysozyme (GH22)
T japonica lysozyme (i-type) (GH22)
Goose egg-white lysozyme (GH23)
Barley chitinase (GH19)
C-end of a-1 helix E22 (general acid) E11 (general acid) E35 (acid–base
residue)
E18 (acid–base residue)
E73 (general acid) E67 (general
acid) b-1 strand E36 (alternative
general base)
base) Loop between b-1
and b2 strands
D40b (general base)
D20b(general base)
b-2 strand T45 (water
positioning)
T26 (water positioning)
D52 (nucleophile)
general base)
S120 (water positioning)
a Nomenclature as in CsnN174 [7] See also Fig 5A b Although localized in the same loop, these two residues are not in equivalent positions when both structures are superimposed [8].
Trang 10position: the DNA of the chitosanase gene harboring a
T45H mutation was obtained in single-stranded form and
hybridized with the oligonucleotide 5¢-CAGAAGCCGATGA
TGGCCGCCNNNGTAGCCCCGGCCGTCACCGATGT-3¢
(N representing any of the four nucleotides inserted at the
positions encoding the 45th residue) This oligonucleotide
harbored also a silent mutation abolishing the sole SacII
restriction site present in the vector After elongation of the
second DNA strand, the resulting double-stranded plasmids
were transformed into E coli BMH 71-18 The
transfor-mants were cultivated overnight in Luria broth with
tetracy-cline Plasmid DNA was isolated and digested with SacII (to
linearize plasmids that did not incorporate the mutagenic
oli-gonucleotide sequence) Plasmid DNA rescued from this
digestion (highly enriched in mutated forms) was
trans-formed into E coli JM109 After selection on tetracycline, a
library of T45-mutated E coli transformants was collected
Mutants of the E36 residue have been produced by a
site-directed mutagenesis method involving PCR using
La Jolla, CA, USA) [35] The procedure was applied to the
csnN174 gene (wild-type or D40G mutant) cloned in the
pUC19 vector in which the E36 codon was localized
between unique restriction sites BamHI and BstXI A first
series of amplifications was performed by using a common
forward primer adjacent to the BamHI (BamHI-F, 5¢-GCT
CACTCATTAGGCACC-3¢) site and the reverse primer for
each specific mutation (E36A-R, 5¢-CCGATGTCCGCGAT
GTACTTG-3¢; E36Q-R, 5¢-CCGATGTCCTGGATGTAC
TTG-3¢) A parallel series of amplifications was performed
by using a common forward primer adjacent to BstXI site
(BstXI-F, 5¢-CTCAGCTGTTGATGAGGT-3¢) and the
for-ward primer for each specific mutation (E36A-F, 5¢-AGTA
CATCGCGGACATCGGTG-3¢; E36Q-F, 5¢-AGTACATC
CAGGACATCGGTG-3¢) After purification of the PCR
products, a second series of PCR was performed with the
same external primers The resulting 1215 bp mutated
frag-ments were cloned between the BamHI and BstXI sites of
pFD-ES vector for expression in S lividans The mutated
DNA sequences were confirmed by DNA sequencing
Revertant selection
Chitosanase-positive revertants after saturation mutagenesis
were selected on toxic chitosan medium Chitosan
acetate buffer pH 5.3 and hydrolyzed with CsnN174
boiled for 30 min to stop the reaction, chilled on ice and
hydrolyzed chitosan was determined using the reducing
sug-ars assay of Lever [36] The toxic chitosan medium was
pre-pared as follows: to a sterile, melted base medium
distilled water), we added (in that order, with constant
gen-tle shaking) sodium citrate buffer (5 mm final
were optimized according to the required level of medium toxicity [22] as described above In some cases,
A mixture of E coli cells, members of the saturation mutagenesis library, was diluted to an approximate cell
colonies were picked up after 48–72 h Revertant charac-terization was completed by sequencing their chitosanase genes
Chitosanase purification and assay
described previously [37] All chitosanase forms were puri-fied from recombinant S lividans TK24 culture super-natants as described previously [10] except that the gel-filtration step was replaced by the more rapid hydroxy-apatite chromatography [15] The CD spectra of the chito-sanase preparations thus obtained were identical to that
of the wild-type enzyme, indicating that the global con-formation was not significantly affected by the individual mutations
Chitosanase assays were performed determined using chitosan Sigma-Aldrich (St Louis, MO, USA) (characterized
sodium acetate buffer (pH 5.5) In standard assay, a
reaction mixtures were set up containing eight different
using microtiter plates Protein concentration and reaction time was adjusted to obtain the same overall hydrolysis level for all studied proteins Reaction time was 10 min for wild-type, E36A, E36D, E36N and E36Q chitosanases, 20 min for the D40G and T45S chitosanases, 50 min for D40G + E36A chitosanase, and for 100 min for D40G + T45D and D40G + E36Q chitosanases Liberation of reducing sugars
were calculated using the non linear least-square fitting pro-cedure for Michaelis–Menten equation in prism software (version 5.0 for Windows, San Diego, CA, USA)
MS set-up and signal correction
Several experiments were performed in continuous-flow mode directly coupled with ESI-MS using a time-of-flight mass spectrometer (Agilent, Santa Clara, CA, USA) The analytical set-up as well as the chosen signal corrections were as published recently [15,38]