Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp490, Glu328, Val327 in OfHex1, and Trp358, Tyr131 and Ile179 in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu328 together with Trp490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K m for (GlcNAc)2 and a 42-fold increment in K i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu368 and Asp367) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu328 and Val327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.


Introduction
Cellulose and chitin, both b-1,4-linked linear saccharides composed of glucose (Glc) or N-acetylglucosamine (GlcNAc), respectively, are the two most abundant biomasses distributed in the plant and animal kingdoms, respectively [1,2]. The biodegradation of these saccharides proceeds via the same path, endoenzymes first degrade higher degree polymerized saccharides into oligosaccharides and then exo-enzymes degrade oligosaccharides into monosaccharides. Cellulase (EC 3.2.1.4) and chitinase (EC 3.2.1.14) are the endo-splitting enzymes, and b-glucosidase (EC 3.2.1.21) and b-N-acetyl-D-hexosaminidase (EC 3.2.1.52) are the exo-splitting enzymes required for cellulose and chitin degradation, respectively [1,2]. The similarity between these two biomolecules hints at a convergent evolution between the degradation enzymes from plants and chitin-containing animals. The recent crystal structural information of exo-splitting enzymes provides evidence of this linkage.
The catalysis proceeds via a double displacement mechanism by which two acidic residues act as nucleophile and acid/base catalyst, respectively [5]. The crystal structure of BGlu1 revealed that BGlu1 possesses a classic (b/a) 8 -barrel catalytic domain constituting a substrate binding pocket with subsites for binding both the leaving Glc (-1 subsite) and the other cellooligosaccharide residues (+1 subsite, +2 subsite and so on) [6,7]. The amino acid residues constituting the subsites for binding the cellooligosaccharide residues, in particular the +1 subsite, are thus determinants for both substrate affinity and specificity. Three residues, Trp 358 , Ile 179 and Tyr 131 , are found to be crucial for the +1 Glc binding. Trp 358 stacks with the +1 Glc through its indolyl group, Ile 179 forms a hydrophobic interaction with the +1 Glc through its isopropyl group, and Tyr 131 forms a hydrogen bond with the C3-OH of the +1 Glc through its phenolic hydroxyl group [6,7]. In this way, the +1 and -1 sugars are stabilized and exist around a 90u dihedral angle adjacent to each other, yielding a conformation where the glycosidic bond between them becomes more susceptible to attack by the catalytic residues.
The structural alignment reveals that the active pocket architecture of OfHex1 and BGlu1 is very similar although their overall topology as well as amino acid sequence shares very little similarity ( Fig. 1) [8]. Insect OfHex1 from the Asian corn borer (Ostrinia furnacalis) is a b-N-acetyl-D-hexosaminidase specialized for chitin degradation during molting and metamorphosis [9]. This enzyme attracts much attention because of its potential as a species-specific target for developing eco-friendly pesticides [10][11][12][13][14][15]. OfHex1 shows high activity toward b-1,4-linked chitooligosaccharides but is not able to hydrolyse b-1,2-linked GlcNAc from N-glycans, thus exhibiting very high substrate specificity [9]. According to the CAZy database [4], b-N-acetyl-D-hexosaminidase belongs to family 20, and uses a substrate-assisted double displacement mechanism by which the 2-acetamido group in the substrate GlcNAc, instead of the acidic residue in a b-glucosidase, acts as the nucleophile [16,17]. OfHex1 also contains a -1 subsite and +1 subsite for sugar moiety binding, and the three corresponding amino acid residues found in BGlu1 for binding the +1 GlcNAc are found in OfHex1(Val 327 , Glu 328 and Trp 490 ).
We thus hypothesized that both cellulolytic and chitinolytic enzymes may possess a similar mechanism to increase their affinities and specificities toward physiological substrates. In this study, a comparative structural investigation between BGlu1 and OfHex1 was performed. Although the two enzymes exhibit no similarity in either their overall structure or amino acid composition of their active pockets, crucial conserved residues were discovered by structure-based sequence alignment among cellulolytic and chitinolytic enzymes from different species. The significance of these residues was investigated by site-directed mutagenesis, biochemical characterization and crystal structure analysis. This research may provide a basis for the development of pesticides that are suitable for use in cases where plant protection is a priority.

Structure Comparison and Multiple Sequence Alignment
Structure comparison was performed by PyMOL (Schrödinger LLC, Portland, OR) and structural figures were also prepared by PyMOL. Multiple sequence alignments were performed using PROMALS3D [18] and the alignment figure was prepared with ESPript [19].
The positive clones were cultured in BMMY broth at 30uC for 144 h, and methanol (1% of the total volume) was added every 24 h. Wild-type and mutant OfHex1 were purified from the culture supernatant by ammonium sulfate precipitation (65% saturation), affinity chromatography on a HisTrap TM HP column (5 ml, GE Healthcare) followed by anion exchange chromatography on a Mono Q TM 5/50 GL column (1 ml, GE Healthcare) [20]. The purity of the mutants was analyzed by SDS-PAGE.
The b-N-acetyl-D-hexosaminidase from Streptomyces plicatus (SpHex) was purchased from New England Biolabs. The b-N-  acetyl-D-hexosaminidase from Serratia marcescens (SmChb) was expressed and purified according to the reported method [21].
For the substrate (GlcNAc) 2 , the reaction mixtures contained 0.04, 0.08, 0.12, 0.16 and 0.2 mM of substrate and an appropriate amount of enzyme in 50 ml of Britton-Robinson's wide range buffer [pH 7.0 for OfHex1, mutants of OfHex1(V327G, E328A, E328Q and W490A) and SmChb, pH 4.0 for SpHex]. For GlcNAcb1,2Man, the reaction mixtures contained 0.03, 0.05, 0.1, 0.2 and 0.4 mM of substrate and an appropriate amount of enzyme in 50 ml of Britton-Robinson's wide range buffer at the same pHs mentioned above. After incubation at 25uC for a specific period, 10 ml of the reaction was immediately analysed using a TSKgel amide-80 column (4.66250 mm, Tosoh) at 25uC. The reaction velocity was quantified by comparing the peak area of the product GlcNAc to a standard curve of GlcNAc at known concentrations. In both cases, the substrate consumption was limited to less than 10%. The K m and k cat values were also calculated by linear regression of the data using Lineweaver-Burk plots.
TMG-chitotriomycin was kindly provided by Professor Biao Yu (Institute of Organic Chemistry, Chinese Academy of Science). The inhibitory kinetics of TMG-chitotriomycin for the wild-type and mutant forms of OfHex1, SmChb and SpHex were studied using 4MU-b-GlcNAc (4-methylumbelliferone-N-acetyl-b-D-glucosaminide, Sigma) as a substrate [8]. The K i values were calculated by linear regression of the data using Dixon plots.

Crystallization and Data Collection
Mutant OfHex1 (E328A) was incubated with excessive TMGchitotriomycin (5-fold the amount of protein), and then concentrated to ,7 mg/ml. Vapour diffusion crystallization experiments were set up at 4uC by mixing 1 ml of protein and 1 ml of mother liquor consisting of 100 mM HEPES (pH 7.4), 200 mM MgCl 2 , and 33% PEG400. Diffraction data of mutant OfHex1 (E328A)-TMG-chitotriomycin complex was collected using an in-house Rigaku Micromax-007 HF (Rigaku Raxis IV++ Image Plate, wavelength 1.5418 Å , at 180 K), and processed using the Crystal Clear software package [22].

Determination and Refinement of Structures
The structure of mutant OfHex1 (E328A)-TMG-chitotriomycin complex was solved by molecular replacement with Molrep [23] using the structure of OfHex1-TMG-chitotriomycin complex (PDB accession number: 3NSN) as the search model. There was one monomer in the asymmetric unit for the structure. Structure refinement was achieved by Refmac5 [24] and CNS [25]. Model building was performed in Coot [26]. The quality of the final model was checked by PROCHECK [27]). All structural figures were prepared by PyMOL.
BGlu1 is a monomeric enzyme with only one catalytic domain while OfHex1 is a dimeric enzyme with an N-terminal zincin-like domain and a C-terminal catalytic domain in each subunit. Although both BGlu1 and OfHex1 possess non-classical catalytic (b/a) 8 -barrels, the overall topology of the two enzymes' catalytic domain is quite different as supported by the fact that the C a atoms of the catalytic domain of BGlu1 (residues 1 to 476) superimposed on those of OfHex1 (residues 207 to 594) gives a RMSD value of 3.8 Å (based on the overlap of 275 C a atoms) by a Dali pairwise comparison (Fig. 1A) [28]. In addition, BGlu1 has four distinguished loops (Loop A, residues 25-65; Loop B, residues 177-206; Loop C, residues 314-363; Loop D, residues 387-403) while OfHex1, in accordance with BGlu1, has two corresponding loops including L 314-335 (residues 314-335) and L 478-496 (residues 478-496).
Overlapping of the +1 subsites in the active pockets of OfHex1 (in blue) in complex with TMG-chitotriomycin (in green) and SmChb (in white) in complex with (GlcNAc) 2 (in pink). Water molecules are shown as red balls and hydrogen bonds are shown as black dashes. The residues of SmChb were grounded. doi:10.1371/journal.pone.0052225.g002 By using the pair-fitting alignment using the atoms in the ring of the -1 sugar (5 carbon atoms and 1 oxygen atom) as fitting pairs, the spatial arrangements of the residues comprising the -1 subsites of BGlu1 and OfHex1 are different. Except for the His 130 /Asn 175 / Glu 176 in BGlu1 and His 303 /Asp 367 /Glu 368 in OfHex1 constituting the catalytic residue clusters, only two pairs of residues were found to be spatially conserved and with similar functions. Trp 433 in BGlu1 and Trp 524 in OfHex1 are located in the bottoms of the -1 subsites and stack with the non-reducing end sugar rings while Gln 29 in BGlu1 and Glu 526 in OfHex1 make hydrogen bonds with C4-OH/C6-OH and C4-OH of -1 sugars, respectively (Fig. S1).
However, a striking similarity in the architectures was discovered in the +1 sugar binding sites (Fig. 1B). Pair-fitting alignment was performed by using the atoms in the ring of the +1 sugar (5 carbon atoms and 1 oxygen atom) as fitting pairs. The spatial locations of interactions for binding the +1 sugar are conserved between the two enzymes.
It is interesting to note that the residues involved in the interactions are located in distinguished loops. Tyr 131 , Ile 179 and Trp 358 in BGlu1 are located in the loop between the b3 strand and the a3 helix, Loop B (between the b4 strand and the a4 helix) and Loop C (between the b6 strand and the a6 helix), respectively [6,7]. Accordingly, both Val 327 and Glu 328 in OfHex1 are located in L 314-335 while Trp 490 is located in L 478-496 [8].
Trp 358 , which stacks with the +1 sugar from the down side in BGlu1, corresponds to Trp 490 in OfHex1. Ile 179 , which forms a hydrophobic interaction with the +1 sugar in BGlu1, corresponds to Val 327 in OfHex1. Moreover, Tyr 131 , which forms a hydrogen bond with the C3-OH of the +1 sugar via a water molecule in BGlu1, corresponds to Glu 328 in OfHex1 which directly interacts with the C3-OH of the +1 sugar. The carboxyl oxygen atom of Glu 328 in OfHex1 is positioned in the same location as a water molecule in BGlu1 which intermediates hydrogen bonding formation between the +1 sugar and BGlu1. One may notice that BGlu1 Glu 440 also makes a water-mediated hydrogen bond with the C3-OH of the +1 sugar, and is almost close enough to make a direct hydrogen bond (3.4 Å in the 3F5K structure), although it also H-bonds to O4 and O6 of the -1 subsite glycosyl residue. However, OfHex1 does not contain such a residue in the active site. Thus, the spatial conservation of the residues Val 327 / Glu 328 /Trp 490 in OfHex1 and Ile 179 /Tyr 131 /Trp 358 in BGlu1 is believed to be functionally important.
The results of the amino acid sequence alignment indicated that, although the length of the loop varies, Trp 490 in the loop (L 478-496 in OfHex1) is highly conserved in all of the selected chitinolytic b-N-acetyl-D-hexosaminidases ( Fig. 2A, Fig. S2). This demonstrated that the stacking interaction between the +1 sugar and the conserved Trp is vital for the function of the chitinolytic b-N-acetyl-D-hexosaminidases. Compared to the loop L 478-496 , the loop L 314-335 varies in both length and amino acid composition, a function of the biodiversity of the species chosen for the sequence alignment ( Fig. 2A).
The residues in the L 314-335 , namely Val 327 and Glu 328 in OfHex1, are also conserved in most b-N-acetyl-D-hexosaminidas-  The structural comparison revealed that the direction of the loop (L 314-335 of OfHex1 and SmChb) is clockwise while that of SpHex is counter-clockwise (Fig. 2B). Although the primary structures of L 314-335 s and L 478-496 s in these enzymes are totally different ( Fig. 2A), both Val 327 and Trp 490 in OfHex1 can be found in SpHex and SmChb (Val 276 and Trp 408 in SpHex, Val 493 and Trp 685 in SmChb) (Fig. 2B). For Glu 328 in OfHex1, SmChb possesses Gln 494 but SpHex does not possess any comparable residues in this position (Fig. 2C). Glu 328 in OfHex1 functions through a hydrogen bond with the C3-OH of the +1 sugar. Gln 494 in SmChb, however, functions the same way as the Tyr 131 in BGlu1, that is, by interacting with the C3-OH of the +1 sugar via a water-mediated hydrogen-bond (Fig. 1A).

Significance of the Residues Conserved in the +1 Subsite
To study the role of Val 327 , Glu 328 and Trp 490 during substrate binding and catalysis, the enzymatic properties of both the wildtype and mutants of OfHex1 were compared by using b1,4conjugated (GlcNAc) 2 and b1,2-conjugated GlcNAcb1,2Man.
Using (GlcNAc) 2 as the substrate, the catalytic efficiency after site-directed mutagenesis was determined (Fig. 3A). The k cat values of the mutants, V327G, E328Q, E328A and W490A were found to be lower than the wild-type, ranging from 12% (E328Q) to 26% (V327G) ( Table 1). The K m values of V327G, E328Q, E328A and W490A mutants were 1-, 2.5-, 8.6-and 12.7-fold higher, respectively, than that of the wild-type. Based on the k cat /K m values, the catalytic efficiencies of the mutants for (GlcNAc) 2 were much lower (from 1.3-to 14.7-fold) than the wild-type. Furthermore, using 4MU-b-GlcNAc as the substrate, the K i values of TMG-chitotriomycin for the wild-type and mutants V327G, E328Q, E328A and W490A were determined ( Table 2). The K i values for TMG-chitotriomycin toward the mutants, V327G, E328Q, E328A and W490A, were increased by 1.1-, 1.6-, 42-and 2277-fold, respectively, compared to the wild-type. It is worthy to note that the mutation of Glu 328 to Ala instead of Gln seriously impaired the ability of OfHex1 to bind the substrate. These results demonstrated that Glu 328 and Trp 490 but not Val 327 are vital for substrate binding.
To better understand the function of Val 327 , additional substrates were studied. As OfHex1 has evolved to degrade b1,4-glycosidic bonds in linear chitooligosaccharides, it is surprising to observe that the mutation of Val 327 to Gly results in OfHex1 having the remarkable capability to hydrolyze the substrate GlcNAcb1,2Man (Table 1 and Fig. 3B). The catalytic efficiency of V327G will be further explored in the discussion section.

Crystal Structure Analysis of E328A Complexed with TMG-chitotriomycin
To reveal the structural basis behind the biochemical data, crystal structural information is needed. Since we have obtained the structure of OfHex1 V327G mutant previously [12], in this study, the crystallization of E328A in complex with TMGchitotriomycin was performed and the complex structure was resolved to 2.5 Å . The statistics of data collection and structure refinement are summarized in Table 3. The coordinates were deposited in the Protein Data Bank with accession number 3VTR. The structure showed well defined unbiased map for TMGchitotriomycin (except GlcNAcIII at the reducing end) and side chains of Asp 367 and Glu 368 (Fig. 4A).
Structure alignment of the OfHex1-TMG-chitotriomycin complex with the E328A-TMG-chitotriomycin complex was performed using PyMOL. According to the alignment, the mutation of Glu 328 to Ala resulted in obvious conformation changes (Fig. 4B). The catalytic residues, Glu 368 and Asp 367 are rotated about 180u and 90u compared to those in the OfHex1-TMGchitotriomycin complex but with similar conformations as those in the apo-structure of OfHex1. The rotation of Glu 368 led to the collapse of the two important hydrogen bond networks (Asp 249 -His 303 -Glu 368 and Glu 328 -H 2 O (I)-Glu 368 ) [8]. In addition, the mutation of Glu 328 to Ala increased the volume of the active pocket from 646.8 Å 3 to 758.3 Å 3 as measured with CASTp [31], suggesting an important role of Glu 328 in constructing the active pocket. The conformation of TMG-chitotriomycin, in particular the GlcNAcII moiety, was found to be very different from that observed in the wild-type (Fig. 4C). Although the sugar rings of GlcNAcII in both complexes are in the 1 C 4 conformation and are superimposable, the C5-hydroxymethyl groups of GlcNAcII orient to different directions.
In addition, the conformation of GlcNAcIII in OfHex1 is O S 2 , but the electron density was not clear enough to identify its conformation in OfHex1 (E328A). However, there is no obvious minus peaks found around GlcNAcIII with the current conformation ( 3,O B) in the difference map. Therefore, this unclearness of the electron density map can be considered as the result of the flexibility of GlcNAcIII.
Furthermore, mutation of Glu 328 to Ala leads to changes in polar interactions between the wild type and the mutant when complexed with TMG-chitotriomycin (Fig. 4C). The most obvious change is the absence of short polar interaction between Glu 328 and the C3-OH of GlcNAcI of TMG-chitotriomycin. Interestingly, a water molecule was found to occupy the position of the side chain of Glu 328 .
Several other changes in intermolecular polar interactions were also observed in the E328A-TMG-chitotriomycin complex when compared to the wild type (Fig. 4C). Firstly, the distance between the nitrogen atom of the 2-acetamido group (GlcNAcI) and the oxygen atom of the carbonyl group (main chain of Val 327 ) increased from 2.8 Å to 3.2 Å . The interaction between the nitrogen atom of the 2-acetamido group (GlcNAcII) and the oxygen atom of the carbonyl group (main chain of Trp 490 ) is missing, but instead, C6-OH of GlcNAcII forms a hydrogen bond with the main chain of Val 327 .

Discussion
Enzymes capable of degrading cellulose and chitin are undoubtedly significant because these saccharides are the two most abundant forms of biomass in nature. Since cellulose and chitin are similar in both their chemical structure and catabolic procedures, it can be deduced that the associated hydrolases would share some commonalities, for example, their substrate binding mechanism or glycosidic bond preference. A structural comparison may provide new clues to uncover the evolutionary divergence of these enzymes between plants and animals as well as provide supporting information for the use of their associated biomass in industrial or medical applications. However, to date, little work has been performed with regard to comparing cellulolytic enzymes with chitinolytic enzymes. Herein, we investigated the structures of a plant cellulolytic enzyme (BGlu1) and an insect chitinolytic enzyme (OfHex1) in an attempt to find their similarities and differences.
A structural comparison revealed that both GH1 BGlu1 and GH20 OfHex1 possess three residues essential for binding the +1 sugar of substrates [6][7][8]. Site-directed mutagenesis, enzyme kinetics as well as crystal structure determination of OfHex1 revealed that Glu 328 and Val 327 are both essential residues for determining glycosidic bond specificity and the +1 sugar binding, respectively.
Site-directed mutagenesis indicated the change of Glu 328 to Ala impaired OfHex1's affinity toward the substrate, (GlcNAc) 2 , by 8fold and the inhibitory activity of TMG-chitotriomycin by 42-fold (Tables 1 & 2). However, the mutation of Glu 328 to Gln only caused a slight impairment in binding (Tables 1 & 2). Thus, we deduced that the impairment is most likely caused by the absence of a strong polar interaction (2.67 Å ) between Glu 328 and GlcNAcI (Fig. 4C).
Moreover, using (GlcNAc) 2 as the substrate, the K m values of SmChb (with Gln 494 corresponding to Glu 328 of OfHex1) is in accordance with that of E328Q, and the K m values of SpHex (no residue at the corresponding spatial location) is in accordance with that of E328A (Table 1). These results further confirmed the involvement of Glu 328 in substrate binding of OfHex1.
Taken all together, since all the chitinolytic enzymes including SmChb, SpHex and OfHex1 contain the conserved Val and Trp residues at their +1 sugar binding sites, the difference in their affinities for (GlcNAc) 2 is most likely because of the presence of Glu 328 (in OfHex1). The appearance of a conserved Glu at the +1 sugar binding sites might be the result of positive evolution.
As indicated by the K m values for cellobiose, BGQ60 has much higher binding affinity at +1 subsite than BGlu1 [32][33][34][35]. But the mutation of Ile 179 to Val does not improve BGlu1's affinity for cellobiose, indicating Ile 179 is not essential for the cellobiose binding [6]. Accordingly, mutation of Val 327 to Gly did not impair OfHex1's affinity for the substrate [(GlcNAc) 2 ] and the inhibitor (TMG-chitotriomycin) ( Tables 1 & 2). These results lead us to believe that OfHex1 Val 327 may not be so crucial for substrate binding ( Fig. 2A).
However, a dramatic conformational change in Val 327 is observed after OfHex1 binds TMG-chitotriomycin [8], namely the isopropyl group of Val 327 moves from a vertical position to a parallel position around the indolyl plane of Trp 490 . As a result, the entrance size of the active pocket is enlarged from 7.25 Å to 8.26 Å . Thus, we assumed that this conformation change may suggest that Val 327 plays some other essential role.
Based on the k cat /K m values, although V327G hydrolyses GlcNAcb1,2Man 3623-fold slower than (GlcNAc) 2 , it is still 9% faster than StrH. StrH is a bacterial b-N-acetyl-D-hexosaminidase from Streptococcus pneumonia that is believed to be able to specifically hydrolyse b1,2-linked GlcNAc substrates [36]. According to the structural comparison of OfHex1 wild-type, V327G and StrH (GH20A-GlcNAcb1,2Man complex), OfHex1 has a narrow active site pocket (Fig. S3A) which constrains the +1 GlcNAc sugar via hydrophobic and polar interactions with Val 327 , Glu 328 and Trp 490 (Fig. 4C). However, StrH (GH20A) has a shallow active pocket (Fig. S3A) in which the +1 Man is almost solvent exposed [37]. Furthermore, as shown in Fig. S3B, the conformations of both the +1 mannose in the StrH complex and the +1 GlcNAc in the OfHex1 complex are 1 C 4 chairs, but the two superimposed sugar ring is separated by a 35.4u dihedral angle [37]. Thus, if GlcNAcb1,2Man binds to the active pocket of OfHex1, steric hindrance might be encountered between the C3-OH and C6-OH of the +1 Man and the isopropyl group of Val 327 . This steric hindrance is removed by the mutation of Val 327 to Gly. Thus, V327G is conferred with the ability to catalyse the hydrolysis of GlcNAcb1,2Man. On the basis of the above findings, Val 327 was speculated to have a role in the b1,4 glycosidic bond specificity of chitinolytic b-N-acetyl-D-hexosaminidases.
In summary, two conserved residues in chitinolytic b-N-acetyl-D-hexosaminidases with totally different functions were discovered by comparative structural analysis between the insect chitinolytic b-N-acetyl-D-hexosaminidase OfHex1 and the plant cellulolytic bglucosidase BGlu1. OfHex1Glu 328 is vital for the binding of chitooligosaccharides while OfHex1Val 327 is responsible for the glycosidic bond specificity. Together with the previously determined role of Trp 490 in binding chitooligosaccharides, the functions of all the three conserved residues comprising the +1 subsite of chitinolytic b-N-acetyl-D-hexosaminidases have been revealed.