Accessory active site residues of Streptomyces sp N174 chitosanase Variations on a common theme in the lysozyme superfamily ` Marie-Eve Lacombe-Harvey1, Tamo Fukamizo2, Julie Gagnon1, Mariana G Ghinet1, Nicole Dennhart3, Thomas Letzel3 and Ryszard Brzezinski1 ´ ´ ´ ´ Departement de Biologie, Centre d’Etude et de Valorisation de la Diversite Microbienne, Universite de Sherbrooke, Canada Department of Advanced Bioscience, Kinki University, Nara, Japan Department for Basic Life Sciences, Technische Universitat Munchen, Freising-Weihenstephan, Germany ă ă Keywords chitinase; chitosanase; glycoside hydrolase; inverting mechanism; lysozyme Correspondence ´ R Brzezinski, Departement 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 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 substrate 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 selection of active chitosanase variants after saturation 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 number 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 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), endohydrolase-type enzymes acting via an inverting mechanism [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 mechanism and are folded similarly, with two globular 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 FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 857 ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase (mostly a-helical) domains separated by a substrate binding cleft [6–9] Site-directed mutagenesis studies of CsnN174, combined 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 eggwhite This group of enzymes is sometimes designated as the lysozyme superfamily [6,8] Despite their structural 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 mechanism with anomeric retention [11] Asp40 of CsnN174 is found inside another element of the central conserved 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 residue with general base function has so far been proposed for goose egg-white lysozyme [12] Details of the catalytic mechanism should vary among these structurally 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 significant 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 population 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 mecha858 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 ⁄ 125 and ⁄ 485 of wild-type, respectively [10] These values were typical of similar mutations in other inverting glycoside hydrolases [13] A mutation path was suggested by Brameld and Goddard to rebuild the GH19 barley inverting chitinase 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 glutamate 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, considering 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 residue 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 previously [10] The data suggested that D40G mutant chitosanase was impaired in its catalytic activity although its substrate-binding mode remained essentially unchanged Table Specific activities and kinetic parameters of purified wildtype and mutant CsnN174 All specific activities were determined at a single chitosan concentration (800 lgỈmL)1) Kinetic parameters were calculated using the non linear least-square fitting procedure for Michaelis–Menten equation in PRISM software v 5.0 ND, not determined Enzyme Specific activity (unitsỈmg)1 protein) Km (lgỈmL)1) kcat (min)1) Wild-type D40G E36A E36Q E36D E36N E36Q + D40G E36A + D40G T45H T45E T45S D40G + T45D D40G + T45E 52.9 1.6 29.2 44.7 29.1 21.9 0.3 0.07 < 0.01 0.011 37.6 0.06 0.02 24.8 22.9 40.2 34.8 25.8 36.1 41.6 52.4 ND 23.5 25.8 31.2 ND 743.9 26.4 688.0 699.8 500.2 409.6 4.1 1.44 ND 2.21 628.9 1.0 ND FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS ` M.-E Lacombe-Harvey et al Interaction of the D40G mutant with the substrate could be investigated in several ways First, the substrate-binding ability was assessed by thermal unfolding experiments (Fig 1) (GlcN)3 binding to the wild-type enzyme increased the transition temperature (Tm) by 5.7 °C, and its binding to D40G increased Tm by 2.8 °C 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 oligosaccharides [5,15,16], however, provides insight into the interaction 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 Active site residues of family 46 chitosanase (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 7.4Ỉmin)1 (wild-type = 385Ỉmin)1) for (GlcN)5 and 3.1Ỉmin)1 (wild-type = 140Ỉmin)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 neither wild-type nor D40G chitosanase are able to cleave GlcNAc–GlcNAc bonds (data not shown) The stereochemistry of the D40G chitosanase reaction 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 molecule was found to attack the C1 carbon of the transition state sugar residue from the side identical to that in the wild-type [3] It was shown recently that a mutation of the general 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 addition of the azide ion The effect of the azide concentration 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 Fig 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 mM sodium acetate buffer pH 5.5 The final concentrations are 2.3 lM for the enzyme and 2.3 mM for the saccharide The unfolding process was monitored by CD at 222 nm 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 FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 859 ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase A B C D Fig 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 mM ammonium acetate-containing aqueous solutions pH 5.2 at 20 °C (A) (GlcN)n hydrolysis time courses obtained for wild-type endochitosanase (5.0 nM) catalyzed reaction performed with 25.0 lM of the substrate (GlcN)5 (100% = 2.3 · 106 counts); (B) (GlcN)n hydrolysis time courses obtained for D40G endochitosanase (200 nM) catalyzed reaction performed with 25.0 lM of the substrate (GlcN)5 (100% = 2.2 · 106 counts); (C) (GlcN)n hydrolysis time courses obtained for wild-type endochitosanase (5.0 nM) catalyzed reaction performed with 25.0 lM of the substrate (GlcN)4 (100% = 2.5 · 106 counts); (D) (GlcN)n hydrolysis time courses obtained for D40G endochitosanase (200 nM) catalyzed reaction performed with 25.0 lM of the substrate (GlcN)4 (100% = 2.8 · 106 counts) (A) and (C) were adapted from Dennhart et al [15] with permission Fig Anomer production from the D40G mutant chitosanase hydrolysis of (GlcN)6 (A) Time-dependent 1H-NMR spectra (B) Time course of anomer production The enzymatic reaction was conducted in 50 mM deuterated sodium acetate buffer pH 5.0 in an NMR tube thermostated at 30 °C because its side chain points towards the substratebinding cleft (Fig 5A) Glu36 appears to be a minor player in the wild-type configuration, because its sub860 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 Km value FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS ` M.-E Lacombe-Harvey et al A Active site residues of family 46 chitosanase 0.12 0.1 M NaN3 0.08 (GIcN)3 (GIcN)6 0.06 0.04 0.02 Concentration (M) 0 40 80 120 160 (GIcN)2 (GIcN)4 200 240 0.12 Fig Chemical rescue experiments (A) Time courses of the enzymatic degradation of (GlcN)6 by D40G in the absence or presence of sodium azide (0.65 and 2.6 M) The enzymatic reaction was conducted in 50 mM sodium acetate buffer pH 5.0 and at 40 °C The enzyme concentration was 8.5 lM Only some examples of tested concentrations are shown (B) Effect of sodium azide concentration 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) 0.1 0.65 M NaN3 (GIcN)3 0.08 (GIcN)6 0.06 A 0.04 (GIcN)2 0.02 (GIcN)4 0 0.12 40 80 120 160 200 (GIcN)3 2.6 M NaN3 0.1 240 0.08 0.06 (GIcN)6 0.04 (GIcN)2 0.02 (GIcN)4 40 80 120 160 200 Reaction time (min) 240 Specific acivity (ì104 MÃmin1ÃàM1) B 0.6 B 0.5 0.4 0.3 0.2 0.1 0 Concentration (mM) C 70 60 50 40 30 20 10 70 60 50 40 30 20 10 1.5 Sodium azide (M) 0.5 M NaN3 2.5 (GIcN)6 (GIcN)3 (GIcN)4 100 200 300 400 500 600 700 2.3 M NaN3 (GIcN)6 (GIcN)3 (GIcN)4 100 200 300 400 500 Reaction time (min) 600 700 Fig (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 Francisco, 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 numbers for GenBank database): BAC_CIRC, B circulans MH-K1 (D10624); BAC-EHIM, Paenibacillus ehimensis EAG1 (AB008788); BUR_GLAD, Burkholderia gladioli (AB029336); BAC_SUBT, Bacillus subtilis (U93875); BAC_AMYL, Bacillus amyloliquefaciens (ABS75305); BAC_KFB, Bacillus sp KFB-CO4 (AF160195); PBCV-1, Chlorella virus of Paramecium bursaria (U42580); CVK2, Chlorella virus CVK2 (D88191); CsnN174, Streptomyces sp N174 (L07779); NOC_N106, Nocardioides sp N106 (L40408); STR_COEL, Streptomyces coelicolor A3(2) (AL109849.1 ORF SC3A3.02) FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 861 ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase (Table 1) The effect of the E36 mutation was quite different in the enzyme with glycine-substituted Asp40 The kcat of 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, putting 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 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 B 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 possible 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 residual 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 similar 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 similar to wild-type This could be confirmed by the reaction time course of D40G + T45D mutant investi862 A Fig Time courses of (GlcN)6 hydrolysis catalyzed by wild-type and D40G + T45D chitosanases monitored by real-time mass spectrometry The enzymatic reactions were carried out in 10 mM ammonium acetate-containing aqueous solutions pH 5.2 at 20 °C (A) (GlcN)n hydrolysis time courses obtained for wild-type endochitosanase (5.0 nM) catalyzed reaction performed with 25.0 lM of the substrate (GlcN)6 (100% = 3.1 · 106 counts); (B) (GlcN)n hydrolysis time courses obtained for D40G + T45D endochitosanase (2.5 lM) catalyzed reaction performed with 25.0 lM of the substrate (GlcN)6 (100% = 3.0 · 106 counts) gated with (GlcN)6 substrate and monitored by realtime MS As shown in Fig 6, even if the enzyme concentration was 500-fold higher in the D40G + T45D reaction (Fig 6B), time course profiles were comparable between the wild-type and double mutant This also indicates a dramatic decrease in enzymatic activity in the double mutant FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS ` M.-E Lacombe-Harvey et al Saturation 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 introduced 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 molecular 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, respectively,  10% and < 0.1% of wild-type activity (I Boucher & R Brzezinski, unpublished data, and Table 1) We used chitosan (0.3 gỈL)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 aqueous solutions than native chitosan A low concentration (5 mm) citrate buffer (pH 6.0) was included in the medium 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 coli strains expressing wild-type or V148T chitosanase 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 chitosanase-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 falsepositive colonies recovered on this medium by mixing various proportions of the V148T chitosanase-expressing cells and chitosanase-negative cells and plating them on media with various salt compositions Both colonies could be distinguished thanks to supplementation with 5-bromo-4-chloro-3-indolyl-b-d-galactopyr- Active site residues of family 46 chitosanase anoside and isopropyl thio-b-d-galactoside, because the empty pAlter-1 vector directs b-galactosidase synthesis 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 mm MgSO4 This salt composition was adopted for the revertant selection experiment After plating the complete T45H-saturation mutagenesis library ( 450 clones) on the optimized chitosan medium, we obtained 55 colonies of putative chitosanase-positive revertants We sequenced csn genes in 15 randomly chosen clones, revealing Thr residues 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 mutagenesis 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 revertants 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 enhancement 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) Analogous 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 FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 863 ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase Gly 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, examination 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 residue 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 resulting from substrate binding 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 difficulties 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 consequences 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 Saturation 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 statistical 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 864 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 residue 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 threonines 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 mechanism 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 positioning 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 primary 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 (residue 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 glycosidases [30] This flexibility results in a variety of conformations for the residues supporting the ‘nucleophilic side’ of the catalytic mechanism observed in the lysozyme superfamily A closer look at the three b-strands segment of the conserved structural core in this superfamily [8] reveals a small number of key structural elements, the residues of which play various functions depending on the enzyme family For example, Glu36 of CsnN174 discussed here lies in a position equivalent FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase Table 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 motifa CsnN174 (GH46) C-end of a-1 helix b-1 strand E36 (alternative general base) D40b (general base) T45 (water positioning) Loop between b-1 and b2 strands b-2 strand Hen egg-white lysozyme (GH22) T japonica lysozyme Goose egg-white (i-type) (GH22) lysozyme (GH23) E35 (acid–base residue) E22 (general acid) E11 (general acid) T4 lysozyme (GH24) E18 (acid–base residue) D30 (nucleophile) E73 (general acid) E67 (general acid) E89 (general base) D20b (general base) T26 (water positioning) D52 (nucleophile) b-3 strand a Barley chitinase (GH19) Nomenclature as in CsnN174 [7] See also Fig 5A positions when both structures are superimposed [8] D97 (putative general base) b S120 (water positioning) Although localized in the same loop, these two residues are not in equivalent to 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 During their evolution from a hypothetical common ancestor, each group of enzymes selected the best positions for essential catalytic residues 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 coli at relatively high molecular mass, whereas chitosan oligosaccharides or short-chain chitosan forms (< 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 transported 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 chitosan This possibility deserves further studies Formal genetic experiments with chitosanase-producing microorganisms are in progress in our group Experimental procedures Bacterial strains and plasmids E coli strains JM109 (endA1, thi, gyrA96, hsdR17 (rk), mk)), relA1, supE44, D(lac-proAB),[F¢, traD36, proAB, laclqzDM15]) and BMH 71-18 (thi, supE, D(lacproAB), [mutS::Tn10] [F¢, proAB, lacIqzDM15]) were used for routine plasmid propagation and as hosts in site-directed mutagenesis procedures of D40G, T45E, T45H, D40G + T45E, T45E + T45D mutants (Promega, Madison, WI, USA) E coli strain DH5a (F) u80lacZDM15 D(lacZYA-argF)U169 recA1, endA1, hsdR17(rk), mk+) phoA, supE44, thi-1, gyrA96, relA1 k)) was used for routine plasmid propagation and as host in site-directed mutagenesis procedures of E36A, E36Q and D40G + E36A Recombinant strains of S lividans TK24 were used for chitosanase production [10] The vector pAlter-1 (for sitedirected 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 derivative 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 intramolecular ligation Site-directed mutagenesis The procedure used to generate mutants D40G, T45E, T45H, D40G + T45E and D40G + T45D has been described previously [10] A variant of this procedure has been used to perform saturation mutagenesis of the T45 FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 865 ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase position: 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 transformants were cultivated overnight in Luria broth with tetracycline Plasmid DNA was isolated and digested with SacII (to linearize plasmids that did not incorporate the mutagenic oligonucleotide sequence) Plasmid DNA rescued from this digestion (highly enriched in mutated forms) was transformed 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 Easy-AÒ High-Fidelity PCR Cloning Enzyme (Stratagene, 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 forward 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 fragments 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 (N-acetylation degree of 21%) was dissolved at 15 gỈL)1 in sodium acetate buffer pH 5.3 and hydrolyzed with CsnN174 (0.025 mL)1) for 10 at 37 °C The hydrolyzate was boiled for 30 to stop the reaction, chilled on ice and lyophilized Number average molecular mass (Mn) of hydrolyzed chitosan was determined using the reducing sugars assay of Lever [36] The toxic chitosan medium was prepared as follows: to a sterile, melted base medium (tryptone; 10 gỈL)1, yeast extract; gỈL)1, agar, 15 gỈL)1 in distilled water), we added (in that order, with constant gen- 866 tle shaking) sodium citrate buffer (5 mm final concentration, pH 6.0), NaCl (200 mm); MgSO4 (3 mm) and chitosan (Mn  15 kDa; 0.3 gỈL)1) Salt and chitosan concentrations were optimized according to the required level of medium toxicity [22] as described above In some cases, 5-bromo-4chloro-3-indolyl-b-d-galactopyranoside (54 lgỈmL)1), isopropyl thio-b-d-galactoside (54 lgỈmL)1) and tetracycline (15 lgỈmL)1) were also included A mixture of E coli cells, members of the saturation mutagenesis library, was diluted to an approximate cell density of · 103ỈmL)1 and plated (100 lLỈplate)1) on chitosan medium Plates were incubated at 37 °C and colonies were picked up after 48–72 h Revertant characterization was completed by sequencing their chitosanase genes Chitosanase purification and assay Chitosanase and protein assays were performed as described previously [37] All chitosanase forms were purified from recombinant S lividans TK24 culture supernatants as described previously [10] except that the gel-filtration step was replaced by the more rapid hydroxyapatite chromatography [15] The CD spectra of the chitosanase preparations thus obtained were identical to that of the wild-type enzyme, indicating that the global conformation was not significantly affected by the individual mutations Chitosanase assays were performed determined using chitosan Sigma-Aldrich (St Louis, MO, USA) (characterized by an N-acetylation degree of 18%) as substrate at 37 °C in sodium acetate buffer (pH 5.5) In standard assay, a concentration of 0.8 mgỈmL)1 was used In kinetic assays, 0.4 mL reaction mixtures were set up containing eight different concentrations (0.02–0.8 mgỈmL)1) of chitosan in eight replicas 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 for wildtype, E36A, E36D, E36N and E36Q chitosanases, 20 for the D40G and T45S chitosanases, 50 for D40G + E36A chitosanase, and for 100 for D40G + T45D and D40G + E36Q chitosanases Liberation of reducing sugars was measured as described previously [37] Km and kcat values were calculated using the non linear least-square fitting procedure 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] FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS ` M.-E Lacombe-Harvey et al Enzymatic assays obtained by real-time MS Batches were prepared in 10 mm ammonium acetate (pH 5.2) aqueous solution containing 5.0 nm wild-type CsnN174 or 200 nm D40G mutant, 1.0 lm malantide (internal standard) and 25.0 lm (GlcN)5 or (GlcN)4, respectively Control experiments were performed using solutions containing 5.0 nm wild-type CsnN174 or 1.0 lm D40G mutant, 1.0 lm internal standard and 25.0 lm (GlcNAc)6 Further batches were prepared in 10 mm ammonium acetate (pH 5.2) aqueous solution containing 5.0 nm wild-type CsnN174 or 2.5 lm D40G + T45D mutant, 1.0 lm internal standard and 25.0 lm (GlcN)6 Several experiments were performed in duplicate Enzyme specific activity was estimated from the slope of the linear portion of degradation curve of the respective oligosaccharides (GlcN)6 or (GlcN)5 substrate during time course analysis Stereochemical course of the enzymatic reaction The substrate (GlcN)6 was lyophilized three times from D2O, and then dissolved in 0.5 mL of 10 mm deuterated sodium acetate buffer, pH 5.0 The substrate solution was placed in a mm NMR tube, and the enzyme (1.5 nmol) added The NMR tube was immediately set into the NMR probe which was thermostatically controled at 30 °C After an appropriate reaction time, accumulation of 1H-NMR spectra was started Each accumulation required The substrate concentration was 8.0 mm Active site residues of family 46 chitosanase raising the temperature, and were > 75% for both proteins tested Chemical rescue experiments The enzymatic reaction of D40G toward (GlcN)6 was monitored by HPLC and refractometric detection in the presence or absence of sodium azide The substrate solutions (72 mm) containing sodium azide were at first prepared with 50 mm sodium acetate buffer, and then each solution pH was adjusted to 4.5 A small amount of D40G enzyme solution was added to each substrate solution, and the reaction mixture was incubated at 40 °C The final concentrations of sodium azide were 0, 0.22, 0.65, 1.6 and 2.6 m, and that of D40G was 8.5 lm When D36A + D40G was used instead of D40G, the final concentrations of the double mutant and sodium azide were 26 lm and 2.3 m, respectively After an appropriate incubation period, a portion of the reaction mixture was withdrawn, and the enzymatic reaction was terminated by mixing with an equal volume of 0.1 m NaOH The resulting solution was applied to the HPLC column of TSK-GEL NH2-60 (Tosoh, 4.6 · 250 mm), eluting with 60% acetonitrile at a flow rate of 0.8 mLỈmin)1 GlcN oligosaccharides were detected with a refractive index monitor, and the individual peak areas in the HPLC profile were converted into molar concentrations using the standard curves obtained by authentic saccharide solutions The molar concentrations of the individual oligosaccharides were plotted against the reaction time to obtain the reaction time course Thermal unfolding experiments Far-UV CD spectra of the chitosanases were obtained in 20 mm sodium phosphate buffer, pH 7.0, using a Jasco J-720 spectropolarimeter (cell length, 0.1 cm) For obtaining thermal unfolding curves of the enzymes, the CD value at 222 nm was monitored while raising the solution temperature at a rate of °CỈmin)1 By setting the thermocouple in the cell, solution temperature was directly measured using a DP-500 thermometer (Rikagaku Kogyo) The same experiments were conducted in the presence of (GlcN)3 in order to examine the stabilization effect caused by the trisaccharide binding to the enzymes To facilitate comparison between the unfolding curves obtained, the experimental data were normalized as follows; fractions of unfolded protein at individual temperatures were calculated from the CD value by linearly extrapolating the preand post-transition baselines into the transition zone, and plotted against temperature Assuming that the unfolding transition of the chitosanase follows a two-state mechanism [39], the unfolding curves obtained by CD were analyzed by least square curve fitting to obtain the midpoint temperatures (Tm) Reversibility values for the unfolding transition were estimated from comparison of the CD value obtained after annealing with that obtained before Reagents Restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA) Chitosan was from SigmaAldrich (St Louis, MO, USA) Chitosan oligosaccharides (GlcN, GlcNn, n = 4–6) and chitin oligosaccharide (GlcNAc6) were purchased from Seikagaku Kogyo Co (Tokyo, Japan) Malantide (internal standard, ‡ 97%) was purchased from Sigma-Aldrich (Steinheim, Germany) Ammonium acetate (> 98%) was obtained from Merck (Darmstadt, Germany), and high-purity water was from a Milli-Q system (Millipore, Eschborn, Germany) All the other reagents and enzyme substrates were of analytical grade and are commercially available Culture media components were obtained from Difco (Mississauga, Canada) Acknowledgements ´ Work at Universite de Sherbrooke was supported by a Discovery grant from the Natural Science and Engi` neering Research Council of Canada to RB M-E L-H is a recipient of a doctoral student fellowship from les ´ Fonds Quebecois de la Recherche sur la Nature et les FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 867 ` M.-E Lacombe-Harvey et al Active site residues of family 46 chitosanase Technologies We thank Isabelle Boucher and Hugo Tremblay for assistance in studies of chitosanase mutants Work at Kinki University was 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chitosanase Biochem Biophys Res Commun 338, 1839–1844 Dennhart N & Letzel T (2006) Mass spectrometric real-time monitoring of enzymatic glycosidic hydrolysis, enzymatic inhibition and enzyme complexes Anal Bioanal Chem 386, 689–698 Honda Y, Fukamizo T, Boucher I & Brzezinski R (1997) Substrate binding to inactive mutants of Streptomyces sp N174 chitosanase: indirect evaluation from the thermal unfolding experiments FEBS Lett 411, 346–350 FEBS Journal 276 (2009) 857–869 ª 2009 The Authors Journal compilation ª 2009 FEBS 869 ... 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... 5-bromo-4-chloro-3-indolyl-b-d-galactopyr- Active site residues of family 46 chitosanase anoside and isopropyl thio-b-d-galactoside, because the empty pAlter-1 vector directs b-galactosidase synthesis in E coli and false... Chitosanase purification and assay Chitosanase and protein assays were performed as described previously [37] All chitosanase forms were purified from recombinant S lividans TK24 culture supernatants