Chitinolytic β-N-acetyl-D-hexosaminidase is a branch of the GH20 (glycoside hydrolase family 20) β-N-acetyl-D-hexosaminidases that is only distributed in insects and micro-organisms, and is therefore a potential target for the action of insecticides. PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate] was initially identified as an inhibitor against GH20 β-N-acetyl-D-hexosaminidases. So far no crystal structure of PUGNAc in complex with any GH20 β-N-acetyl-D-hexosaminidase has been reported. We show in the present study that the sensitivities of chitinolytic β-N-acetyl-D-hexosaminidases towards PUGNAc can vary by 100-fold, with the order being OfHex1 (Ostrinia furnacalis β-N-acetyl-D-hexosaminidase)<SmCHB (Serratia marcescens chitobiase)<SpHex (Streptomyces plicatus β-N-acetyl-D-hexosaminidase). To explain this difference, the crystal structures of wild-type OfHex1 as well as mutant OfHex1(V327G) in complex with PUGNAc were determined at 2.0 Å (1 Å=0.1 nm) and 2.3 Å resolutions and aligned with the complex structures of SpHex and SmCHB. The results showed that the sensitivities of these enzymes to PUGNAc were determined by the active pocket size, with OfHex1 having the largest but narrowest entrance, whereas SpHex has the smallest entrance, suitable for holding the inhibitor, and SmCHB has the widest entrance. By widening the size of the active pocket entrance of OfHex1 through replacing the active site Val327 with a glycine residue, the sensitivity of OfHex1 to PUGNAc became similar to that of SmCHB. The structural differences among chitinolytic β-N-acetyl-D-hexosaminidases leading to different sensitivities to PUGNAc may be useful for developing species-specific pesticides and bactericides.
- O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate (PUGNAc)
The importance of β-N-acetyl-D-hexosaminidase (EC 18.104.22.168) lies in the enzyme's ability to liberate terminal GlcNAc or GalNAc from the non-reducing ends of a variety of saccharides, including oligosaccharides, glycoproteins and glycolipids. According to the CAZy database [1,2], β-N-acetyl-D-hexosaminidase belongs to GH3 (glycosyl hydrolase family 3), GH20 and GH84. Both GH20 and GH84 β-N-acetyl-D-hexosaminidases use a classical substrate-assisted mechanism in which the carbonyl oxygen of the 2-acetamido group of substrate acts as a catalytic nucleophile that attacks the anomeric C1 to form an oxazoline intermediate. On the contrary, GH3 β-N-acetyl-D-glucosaminidase uses the classic double-displacement mechanism in which an enzymatic carboxylate group acts as a catalytic nucleophile to form a covalent glycosyl-enzyme intermediate. Compared with GH3 and GH84 members, GH20 β-N-acetyl-D-hexosaminidases are more complex in terms of structures and physiological functions. GH20 enzymes normally exist in the form of a dimer, fulfilling various physiological roles, such as chitin degradation, N-glycan modification, glycoconjugate degradation and egg–sperm interaction .
Chitinolytic β-N-acetyl-D-hexosaminidases are a branch of GH20 β-N-acetyl-D-hexosaminidases that exclusively function in the degradation of chitin, the linear polymer of β-1,4-linked GlcNAc. In insects, chitin is a structural component of cuticle and peritrophic membranes. Insect chitinolytic β-N-acetyl-D-hexosaminidase has been applied to degrade old chitinous cuticles during metamorphosis [4,5]. In bacteria and fungi, this enzyme is required for nutritious purposes because chitin can be utilized as a source of nitrogen and carbon . Since chitin is absent in higher organisms (plants and vertebrates), chitinolytic β-N-acetyl-D-hexosaminidase has become a potential target for developing pesticides and bactericides.
PUGNAc, chemically formulated as O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate (Figure 1A), was first synthesized as an inhibitor against GH20 β-N-acetyl-D-hexosaminidases, including those from cattle (Bos taurus), plant (Canavalia ensformis) and fungus (Mucor rouxii) . It is an N-acetylglucosaminono-1,5-lactone derivative, which was previously thought to adopt a conformation similar to a glycopyranosyl cation as a transition-state analogue. So far, crystal structures of PUGNAc in complex with GH3 and GH84 enzymes have been published by van Aalten and co-workers  and Vacadlo and co-workers . However, although PUGNAc was first identified as an inhibitor of GH20 β-N-acetyl-D-hexosaminidases, no crystal structure of PUGNAc in complex with a member of this family has been reported.
We show in the present study that the Ki values of PUGNAc for GH20 enzymes varied, and also noticed that, within GH20 chitinolytic enzymes, the Ki values of PUGNAc was in the range of 1 nM for the bacterial enzyme to 102 nM for the insect enzyme. OfHex1 is an insect chitinolytic GH20 β-N-acetyl-D-hexosaminidase from the destructive pest Ostrinia furnacalis . Our previous work has revealed the structural determinants that distinguish chitinolytic GH20 β-N-acetyl-D-hexosaminidases from human lysosomal β-N-acetyl-D-hexosaminidases (HsHexA/HsHexB) . The representative chitinolytic enzymes, OfHex1 and SmCHB (Serratia marcescens chitobiase) , have a deeper substrate-binding pocket that enables them to bind long- and linear-chained substrates such as chito-oligosaccharides, whereas lysosomal enzymes such as HsHexA/B have a shallower pocket that binds branching N-glycans and glycolipids [13,14]. However, the large difference in Ki values of PUGNAc for these enzymes was unexpected. In order to explain this phenomenon, we determined the crystal structures of wild-type as well as the mutant OfHex1(V327G) in complex with PUGNAc at 2.0 Å (1 Å=0.1 nm) and 2.3 Å resolutions and compared their active pocket sizes with the structures of known GH20 chitinolytic enzyme complexes, and found that a subtle difference in the enzyme active pocket size was the key to the different sensitivities of these enzymes towards PUGNAc. The present study not only explains the inhibition mechanism of PUGNAc against GH20 enzymes, but can also provide useful information for the development of specific and potent pesticides derived from PUGNAc.
MATERIALS AND METHODS
Preparation of GH20 OfHex1, SpHex (Streptomyces plicatus β-N-acetyl-D-hexosaminidase) and SmCHB
OfHex1 and its mutants (W490A and V327G) were expressed in Pichia pastoris strains . The molecular masses of monomers were approximately 66 kDa. Single amino acid mutations of OfHex1 were made from the wild-type OfHex1 construct by PCR-based site-directed mutagenesis using the following primers: OfHex(W490A), forward primer, 5′-GCTTGTTCTCCTTACATCGGATGGCAG-3′ and reverse primer, 5′-GATGTAAGGAGAACAAGCGTTGTTACCAGC-3′; OfHex(V327G), forward primer, 5′-GGTGAGCCCCCATGCGGTCAGCTC-3′ and reverse primer, 5′-CGCATGGGGGCTCACCGCAGTATGATTTCC-3′. The recombinant plasmids were linearized and transformed into GS115 cells. The transformants were selected and cultured as described previously . OfHex1 wild-type and mutants were purified from the culture supernatant by ammonium sulfate precipitation (65% saturation), followed by metal chelate chromatography on an IMAC (immobilized metal-ion-affinity chromatography) Sepharose High Performance column (GE Healthcare) and anion-exchange chromatography on a Q Sepharose High Performance column (GE Healthcare) . SpHex was purchased from New England Biolabs. SmCHB was purified from the periplasmic proteins produced by a recombinant Escherichia coli as described previously .
PUGNAc was purchased from Toronto Research Chemicals. TMG-chitotriomycin (Figure 1B) was provided by Professor Biao Yu (Institute of Organic Chemistry, Chinese Academy of Science, Shanghai, China). All inhibition constants for PUGNAc and TMG-chitotriomycin against GH20 enzymes were measured at 25 °C using pNP-β-GlcNAc (4-nitrophenyl-N-acetyl-β-D-glucosaminide; Sigma) as substrate. OfHex1 wild-type and mutants were assayed in 50 mM sodium phosphate buffer (pH 7.0). SpHex and SmCHB were assayed in 50 mM sodium citrate buffer (pH 4.0) and 50 mM sodium phosphate buffer (pH 8.0) respectively. After incubating for 5 min, 0.5 M Na2CO3 was added to the reaction mixture to stop the reaction and the absorbance at 405 nm was monitored using a Sunrise microplate reader (TECAN). The Ki values were determined by steady-state kinetics. The substrate concentrations were 0.2 mM and 0.5 mM. The concentrations of PUGNAc and TMG-chitotriomycin varied for different enzymes. The Ki values were calculated by linear regression of data in Dixon plots.
Crystallization and data collection
Wild-type OfHex1 and OfHex1(V327G) were incubated with excess PUGNAc (5-fold the amount of protein), and then concentrated to ~7 mg/ml. Vapour diffusion crystallization experiments were set up at 4 °C by mixing 1 μl of protein and 1 μl of mother liquor consisting of 100 mM Hepes (pH 7.0), 200 mM MgCl2 and 30% PEG [poly(ethylene glycol)] 400 for OfHex1–PUGNAc, and 100 mM Hepes (pH 7.0), 200 mM MgCl2 and 25% PEG 400 for OfHex1(V327G)–PUGNAc. Diffraction data of wild-type OfHex1–PUGNAc was collected at the Shanghai Synchrotron Radiation Facility, BL-17U [MARMOSAIC 225 mm CCD (charge-coupled-device), wavelength 0.9718 Å, at 100 K], and processed using the HKL2000 package . Diffraction data of OfHex1(V327G)–PUGNAc was collected with in-house Rigaku Micromax-007 HF (Rigaku Raxis IV++ Image Plate, wavelength 1.5418 Å, at 100 K), and processed using the Crystal Clear software package.
Determination and refinement of structures
The structures of wild-type OfHex1–PUGNAc and OfHex1(V327G)–PUGNAc were solved by molecular replacement with MolRep  using the native OfHex1 structure (PDB code 3NSM) as the search model. There was one monomer in the asymmetric unit for each structure. Structure refinement was achieved by Refmac5  and CNS . Model building was performed in Coot . The quality of the final model was checked by PROCHECK . All structural Figures were prepared using PyMOL (DeLano Scientific, http://www.pymol.org).
Inhibitory activities of PUGNAc and TMG-chitiotriomycin
PUGNAc is known as a broad-spectrum inhibitor of glycoside hydrolases [7–9,23], and has been tested against several GH20 β-N-acetyl-D-hexosaminidases, including HsHexB and BtHex (Bos taurus β-N-acetyl-D-hexosaminidase) (mammal), CeHex (Canavalia ensformis β-N-acetyl-D-hexosaminidase) (plant) and MrHex (Mucor rouxii β-N-acetyl-D-hexosaminidase) (fungus). The Ki values of PUGNAc for HsHexB, BtHex, CeHex and MrHex are 36, 110, 100 and 40 nM respectively [7,8]. Since β-N-acetyl-D-hexosaminidases encoded by different genes appear to have different and specific functions, we hypothesized that these functions are conferred by the differences in the structures of the enzymes. In the present study we tested three enzymes that were supposed to function differently from mammalian or plant enzymes, and these were the insect (OfHex1) and two bacterial (SpHex and SmCHB) enzymes. OfHex1 and SmCHB are known for their roles in chitin degradation [10–12], whereas the physiological role of SpHex has not been conclusively established .
Using pNP-β-GlcNAc as substrate, the inhibition of OfHex1, SpHex and SmCHB by PUGNAc was determined to be competitive (Figure 2 and Table 1). Ki of the OfHex1(V327G) to PUGNAc was also investigated because Val327 localizing at the active pocket entrance of OfHex1 is thought to be responsible for substrate binding. To our surprise, PUGNAc acted as a highly efficient inhibitor against SpHex, with a Ki of 0.003 μM. However, it exhibited moderate activity against SmCHB and OfHex1(V327G), with Ki values of 0.058 μM and 0.045 μM respectively, but was less active towards OfHex1, with a Ki of 0.24 μM. In contrast, inhibition of these enzymes by the previously discovered selective inhibitor TMG-chitotriomycin [25,26] yielded a Ki value of 0.065 μM for OfHex1 , which is 16-fold lower than the Ki value for SpHex  but the same (0.077 μM) for both SmCHB and OfHex1(V327G) (Table 1). Compared with TMG-chitotriomycin, PUGNAc is composed of a terminal GlcNAc residue, an oxime group and a phenylcarbamate group instead of a sterically large N,N,N-tri-methyl-D-glucosamine residue conjugated by three GlcNAc residues found in TMG-chitotriomycin.
The order of sensitivity to PUGNAc for GH20 enzymes was SpHex>SmCHB [OfHex1(V327G)]>HsHexB (bovine BsHex)>OfHex1, whereas the order of sensitivity to TMG-chitotriomycin was reversed, namely OfHex1>SmCHB [OfHex1 (V327G)]>SpHex. In our previous study, TMG-chitotriomycin exhibited no activity towards CeHex and HsHexB . Also, widening the active-pocket entrance by the site mutation V327G in OfHex1 dramatically changed the inhibitory properties of OfHex1 to both PUGNAc and TMG-chitotriomycin (Table 1), Taken together, GH20 β-N-acetyl-D-hexosaminidases appeared to vary in biochemical properties in addition to physiological functions, and structural discrepancy may account for the difference.
Structure of OfHex1 in complex with PUGNAc
To understand the structural differences that affect the sensitivities of GH20 enzymes toward PUGNAc, PUGNAc was co-crystallized with OfHex1 and resolved at 2.0 Å. The statistics of the data collection and structure refinement are summarized in Table 2. The co-ordinates of the OfHex1–PUGNAc complex were deposited in the Protein Data Bank (accession number 3OZP) and the structure is shown in Figure 3. The enzyme appeared as a homodimer with each subunit containing one zincin-like domain and one catalytic (β/α)8-barrel domain that bears the active pocket. As many as four tryptophan residues (Trp424, Trp448, Trp490 and Trp524) and one tyrosine residue (Tyr475) constitute a hydrophilic pocket, with a narrow entrance guarded by the vertical indolyl group of Trp490 and the isopropyl group of Val327. Three indolyl groups, one each from Trp424, Trp448 and Trp490, constitute three sides of the pocket wall, and two acidic side chains of the catalytic residues, Asp367 and Glu368, constitute the remaining side. The indolyl group of Trp524 forms the bottom of the pocket.
PUGNAc is bound to OfHex1 with its GlcNAc residue accompanied by the hydrophobic aromatic groups at the −1 subsite of the active pocket, whereas its oxime and phenylcarbamate groups interact with the side chain of Trp490, which lies at the entrance of the pocket (Figure 3A). The C-3, C-4 and C-6 hydroxy groups of GlcNAc were hydrogen bonded to Arg220, Asp477, Glu526 and Trp490 at the active pocket (Figure 3C). The pyranose ring of GlcNAc was in planar 4E envelope conformation and its C-1 atom was in the same sp2 hybridization as reported for GH3 and GH84 enzymes [8,9]. 4E envelope conformation of GlcNAc has been found in the crystal structures of PUGNAc–GH3 VcNagZ  and GH84 CpNagJ , and this indicates that the general inhibitor PUGNAc may inhibit GH3, GH20 and GH84 β-N-acetyl-D-hexosaminidases through a similar inhibition mechanism by mimicking the common oxocarbenium ion-like transition state.
The carbonyl oxygen of the 2-acetamido group in GlcNAc is orientated towards the C-1 atom of the same GlcNAc by interacting with Asp367, Trp448 and Tyr475 (Figure 3C). According to the substrate-assisted catalytic mechanism that is used by GH3, GH20 and GH84 enzymes, the 2-acetamido oxygen atom would act as a catalytic nucleophile and attack C-1 to form an oxazolinium intermediate. Thus it is not surprising that the localization of the 2-acetamido group is highly conserved in other crystal complexes, including PUGNAc–GH84 CpNagJ , chitobiose–GH20 SmCHB  and GalNAc-isofagomine–GH20 HsHexB .
The other part of PUGNAc, which comprises one oxime group and one phenylcarbamate group, is extended to the outside of the pocket through the pocket entrance, comprising Val327 and Trp490. Relative to the oxime plane, the phenylcarbamate group is rotated counterclockwise 66.1° around the ether oxygen atom. The oxime group is sandwiched between the side chains of Trp448 and Glu368, and the phenylcarbamate group is exposed to solvent (Figure 3C). Distortion of PUGNAc is also noticeable. The phenyl plane in the phenylcarbamate group is rotated 81.1° around the plane of its peptide bond. However, in the crystal structures of PUGNAc–GH3 VcNagZ (Ki=0.048 μM)  and GH84 CpNagJ (Ki=0.005 μM) , these two planes are with dihedrals of 2.2° and 34.6° respectively, meaning a relaxed configuration of PUGNAc, leading to higher inhibitory activities (Figure 3E). Since no interaction leading to the stabilization of the phenyl group was observed, we could not understand how this could happen. The most plausible explanation could be that the rotation is the result of balancing energy sustained by an unstablized carbamate group.
Structure of OfHex1(V327G) in complex with PUGNAc
To understand the structural basis of the Ki of PUGNAc for OfHex1(V327G) being 5.3-fold lower than that for the wild-type OfHex1, PUGNAc was co-crystallized with OfHex1(V327G) and resolved at 2.3 Å resolution. The statistics of the data collection and structure refinement are summarized in Table 2. The co-ordinates of OfHex1(V327G)–PUGNAc were deposited in the Protein Data Bank (accession number 3S6T).
As expected, in the OfHex1(V327G)–PUGNAc complex, the isopropyl group of Val327 is replaced by a sterically small hydrogen atom, which leads to a widened active-pocket entrance (Figure 3B). Structural alignment of the wild-type OfHex1–PUGNAc complex with the OfHex1(V327G)–PUGNAc complex suggests that the four tryptophan residues Trp424, Trp448, Trp490 and Trp524 as well as the tyrosine residue Tyr475 are superimposed, and interactions between the GlcNAc moiety of PUGNAc are reproduced (Figure 3D). Also, as seen for the wild-type, the phenylcarbamate group is left exposed to solvent. The conformation of the catalytic Glu368 is almost the same as in the apo-OfHex1 and the putative polar interactions between Glu368 with nitrogen in the oxime group of PUGNAc is absent.
A noticeable difference of PUGNAc's conformations between wild-type and mutant OfHex1 is that the phenylcarbamate group of PUGNAc is only rotated 8.2° around the ether oxygen atom relative to the oxime plane (66.1° in the wild-type) and the dihedral between the phenyl plane and the peptide bond plane is reduced to 59.3° (81.1° in the wild-type) (Figure 3E). This change lets PUGNAc position in a more sterically eased state, meaning PUGNAc has lower binding energy. In addition, the phenyl group of PUGNAc seems to stack better with Trp490 of mutant OfHex1. On the basis of the above observations, we believe the widened active-pocket entrance is the key for higher sensitivity of the OfHex1(V327G) over the wild-type OfHex1 towards PUGNAc.
OfHex1 containing a flexible-sized active pocket
The bacterial GH20 β-N-acetyl-D-hexosaminidases SpHex [28,29] and SmCHB  are known for their relatively rigid active pockets as revealed by the structural differences between the free enzymes and enzymes bound to inhibitors. However, for the insect GH20 OfHex1, large conformational changes in the active site residues were observed when OfHex1 was bound to TMG-chitotriomycin .
To investigate these conformational changes and the flexibility of the active pocket, the structures of apo-OfHex1 and OfHex1–PUGNAc were superimposed. As shown in Figure 3(D), the same conformational changes in catalytic residues were observed for the enzyme in complex with PUGNAc as in the case of the enzyme–TMG–chitotriomycin complex. The two catalytic residues Glu368 and Asp367 are rotated approximately 180° and 90° respectively. Two other residues, His303 and Trp448, are rotated approximately 30° and 45° respectively. Since the ‘lid’ residues Glu368 and Trp448 are rotated at approximately the same magnitude as that in the OfHex1–TMG-chitotriomycin complex, it infers that the ‘open-close’ mechanism is also present in the OfHex1–PUGNAc complex (Figure 3F). This is similar to the OfHex1(V327G)–PUGNAc complex, except Glu368. Glu368 does not rotate but remains in the same comformation as it is in the apo-OfHex1, meaning that Glu368 has a flexible side chain to target the 2-acetamido group of substrate. Thus it can be concluded that OfHex1 has a flexible active pocket.
Our previous work explained how chitinolytic β-N-acetyl-D-hexosaminidase might carry out the degradation of chitin, and provided the structural evidence of what determines the specialized physiological functions of human and bacterial GH20 β-N-acetyl-D-hexosaminidases . However, the differences in enzyme structures responsible for the large difference in Ki values of PUGNAc were undetermined.
In the present study we investigated the active site of the insect β-N-acetyl-D-hexosaminidases OfHex1 by comparing it with those of the bacterial enzymes SmCHB and SpHex, as well as mutant OfHex1(V327G). Even though both key residues and active site architectures are conserved, subtle differences in structures of the active pockets can still be seen and may account for the differences in the sensitivities of these enzymes towards PUGNAc.
Residue localization is highly conserved
To explain why the inhibition of GH20 β-N-acetyl-D-hexosaminidases by PUGNAc gave different Ki values ranging from 1 to 102 nM, the sequences encoding the (β/α)8-barrel catalytic domains of OfHex1 , SpHex (PDB code 1M01) [28,29] and SmCHB (PDB code 1QBB)  as well as the topologies of these three enzymes were analysed. The overall identity among these enzymes is rather low; ~30% between SmCHB and SpHex, 22% between OfHex1 and SpHex, and as low as 17% between SmCHB and OfHex1. On the other hand, a high degree of conservation in the overall topology of the catalytic domains and the localizations of key residues indicate that the key residues still occupy the same positions within the active pockets of these enzymes. These key residues include the catalytic residues (aspartate, histidine and glutamate in the loop between β4 and α4), residues for stabilizing the hydroxy groups of C-3 (arginine in the loop between β1 and α1), C-4 (aspartate in the loop between β7 and β8) and C-6 (glutamate in the loop between β8 and α8) in the pyranose ring, as well as the hydrophobic residues comprising the wall (valine in the loop between β3 and α3, tryptophan in strand β5, tryptophan in the loop between β6 and α6, tyrosine and tryptophan in the loop between β7 and β8) and bottom (tryptophan in the loop between β8 and α8) of the active pocket (see Supplementary Figure S1 and Table S1 at http://www.BiochemJ.org/bj/438/bj4380467add.htm).
Interactions within the active pocket are different
On the basis of the crystal structures, different interactions are responsible for the stabilization of the active pockets of OfHex1 , SpHex [28,29] and SmCHB . Three conserved tryptophan residues that comprise the active pocket wall are stabilized by hydrogen bonds in different manners as far as hydrogen-bond length is concerned.
In OfHex1, the bond between β5 Trp424 and β3 Glu297 is 3.09 Å, and between β6 Trp448 and Thr425 (in the L425–426) was 2.97 Å. Trp490 is less flexible, although it seems to have no obvious interaction with other residues (Figure 4A).
In SpHex, β5 Trp344 is connected to β3 Glu244 by a 3.02 Å bond, whereas β6 Trp361 is connected to α5 Gln346 by a 2.85 Å bond, and Trp408 is connected to Arg365 by a 2.82 Å bond (Figure 4B).
In SmCHB, β5 Trp616 is connected to the β3 Glu446 by a 2.82 Å bond and β6 Trp639 is connected to α5 Asp618 by a 2.89 Å bond. Also, the bond between Gln617 and Asp640 supports the interaction between Asp618 and Trp638. Although similar to Trp490 in OfHex1, Trp685 in SmCHB is not stabilized by any type of interaction, but is sterically restricted by Gly686, which is hydrogen bonded to Tyr643 (Figure 4C).
Active pocket size determines selectivity toward PUGNAc
As mentioned above, the active pocket of GH20 β-N-acetyl-D-hexosaminidase is composed of three catalytic acidic residues (histidine, aspartate and glutamate), six hydrophobic residues (one valine residue, four tryptophan residues and one tyrosine residue) and three additional residues (arginine, aspartate and glutamate) for stabilizing the pyranose ring of GlcNAc (Supplementary Table S1). Although the locations of these residues are conserved and all interactions are within the active pocket, we observed that the sizes of the active pockets vary among different GH20 β-N-acetyl-D-hexosaminidases.
To compare the sizes of the active pockets, the ligand size was considered. Since PUGNAc contains a GlcNAc that is localized in the active pocket upon binding with the enzyme, we chose the structure of the complex that contains a ligand with a GlcNAc component. The volume of the active pockets (comprised of 12 conserved residues, Supplementary Table S1) of OfHex1–PUGNAc, SpHex–GlcNAc [28,29] and SmCHB–chitobiose , were calculated using the software CASTp (http://sts.bioengr.uic.edu/castp/) . Thus the volume of the active pockets was arrayed in the order OfHex1 (333.4 Å2)>SmCHB (291.6 Å2)>SpHex (258.7 Å2). It is interesting to note that the order of volume is in good agreement with that of the Ki values of PUGNAc.
To find the amino acid residues responsible for differences in active pocket sizes, the crystal structure of the OfHex1–PUGNAc complex was superimposed with the SmCHB–chitobiose complex and the SpHex–GlcNAc complex.
As for SmCHB, good superimposition was obtained for the two enzymes, except for the ‘lid’ residues at the active pocket entrances: tryptophan (Trp448 in OfHex1), valine (Val327 in OfHex1, Val493 in SmCHB) and tryptophan (Trp490 in OfHex1, Trp685 in SmCHB) (Figure 4D). Compared with that of Trp448 in OfHex1, the distance between the indolyl group of Trp685 and the two catalytic residues of OfHex1, Asp367 and Glu368, are 1.03 Å and 0.28 Å respectively shorter than the corresponding catalytic residues Asp539 and Glu540 of SmCHB. These shortened distances probably make the pocket more compact when accommodating the 2-acetamido group. On the other hand, the structural difference between OfHex1 and SmCHB presented by valine and tryptophan at the pocket entrance is a narrower entrance (7.13 Å) in the case OfHex1, and a wider entrance (7.94 Å) in the case of SmCHB. Altering the entrance size by mutating Val327 in OfHex1 to glycine resulted in the Ki values of PUGNAc and TMG-chitiotriomycin being almost equal to the Ki values for SmCHB, suggesting that the entrance size may be the key factor that distinguishes OfHex1 from SmCHB (Table 1). The crystal structure of OfHex1(V327G) shows how a site mutation that widens the active-pocket entrance changes PUGNAc's configuration from a tense to an eased state and, in turn, changes Ki.
However, the active pocket of SpHex does not superimpose well with that of OfHex1, but is more likely to be wrapped by that of OfHex1. The positions of residues involved in catalysis (Asp367 and Glu368 in OfHex1, Asp313 and Glu314 in SpHex) and polar interactions are very conserved (Arg211, Asp477, Glu526 in OfHex1; Arg162, Asp395 and Glu444 in SpHex) (Figure 4E). However, the distances between the catalytic residues and the aromatic residues comprising the active pocket wall are different. For SpHex, these differences include 0.74 Å shorter for Trp344 (Trp424 in OfHex1), 1.45 Å shorter for Trp361 (Trp448 in OfHex1), 0.91 Å shorter for Tyr393 (Tyr475 in OfHex1) and 1.73 Å shorter for Trp408 (Trp490 in OfHex1). It is worthy to note that the distance between the conserved residues, valine and tryptophan, at the entrance of the active pocket, is another restriction factor that determines species-specificity. Val276 in SpHex is rotated approximately 60° relative to the position of Val327 in OfHex1, and is positioned 2.61 Å away from the active pocket centre, causing the isopropyl group of Val327 in OfHex1 to hinder the phenylcarbamate group of PUGNAc approaching the catalytic Glu368. Mutation of Val327 to glycine caused a 5.3-fold decrease in the Ki value of PUGNAc for OfHex1, but was still 15-fold higher than that for SpHex (Table 1).
Thus the order of susceptibility of these enzymes towards TMG-chitotriomycin and PUGNAc can now be understood, and this also explains the differences in Ki values of PUGNAc for these enzymes in mechanistic terms. Taken together, we concluded that OfHex1 has the biggest active pocket but the narrowest entrance, whereas SpHex has the smallest active pocket, and such three-dimensional structural differences may not have been fully exploited in drug discovery, but could well be responsible for OfHex1's preference towards a larger ligand, e.g. a GlcNAc derivative with bulky substitutes such as TMG-chitotriomycin, or SpHex's preference towards a smaller ligand, e.g. PUGNAc. The present study should help with the design of highly selective pesticides or bactericides that are derived from PUGNAc.
Tian Liu and Qing Yang designed the research. Tian Liu, Haitao Zhang, Fengyi Liu and Lei Chen performed the research. Tian Liu, Haitao Zhang and Qing Yang analysed data, and Tian Liu, Qing Yang and Xu Shen wrote the paper.
This work was supported by the National Key Project for Basic Research [grant number 2010CB126100], the National Natural Science Foundation of China [grant number 31070715], the National High Technology Research and Development Program of China [grant number 2011AA10A204], the National Key Technology R&D Program [grant number 2011BAE06B05] and the Fundamental Research Funds for the Central Universities [grant number DUT11ZD113].
We thank all of the staff at the beamline BL17U at the Shanghai Synchrotron Radiation Facility (China). We thank Dr Alan K. Chang (Dalian University of Technology) for his contribution in the language editing of the paper prior to submission.
The crystal structures of OfHex1–PUGNAc complex and mutant OfHex1(V327G)–PUGNAc complex have been deposited in the Protein Data Bank (PDB) with accession numbers 3OZP and 3S6T.
Abbreviations: BtHex, Bos taurus β-N-acetyl-D-hexosaminidase; CeHex, Canavalia ensformis β-N-acetyl-D-hexosaminidase; GH, glycoside hydrolase; HsHex, human β-N-acetyl-D-hexosaminidase; MrHex, Mucor rouxii β-N-acetyl-D-hexosaminidase; OfHex1, Ostrinia furnacalis β-N-acetyl-D-hexosaminidase; PEG, poly(ethylene glycol); pNP-β-GlcNAc, 4-nitrophenyl-N-acetyl-β-D-glucosaminide; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate; SmCHB, Serratia marcescens chitobiase; SpHex, Streptomyces plicatus β-N-acetyl-D-hexosaminidase
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