Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases

Abstract

Endo-beta-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (K(d) = 28 microm) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

FIGURE 1.

The reaction catalyzed by GH85 endo-β-glucosaminidases including Endo-D and substrates and inhibitors used in this study. A, reaction catalyzed by GH85 enzymes cleaves the chitobiose core of N-glycans to generate a free N-glycan bearing a single GlcNAc residue at the terminus, and the liberated protein in which a single GlcNAc residue is N-linked to the protein. The glycosidic bond cleaved is indicated with an arrow. B, Structure of the oxazoline intermediate proposed for glycoside hydrolases using a substrate assisted catalytic mechanism. C, structures of the series of β-glucosaminide substrates used in this study that have varying degrees of fluorination in the acyl group. D, structure of NAG-thiazoline.

FIGURE 2.

Kinetic studies of SpGH85. A, pH dependence of V_max/K_m. Full Michaelis-Menten parameters were determined at each pH using 3F4NP-GlcNAc as substrate. B, initial velocity of the SpGH85-catalyzed hydrolysis of N-fluoroacetyl derivatives of 3F4NP-GlcNAc-F₀ (○)(1a); 3F4NP-GlcNAc-F₁ (•)(1b), 3F4NP-GlcNAc-F₂ (□) (1c), and 3F4NP-GlcNAc-F₃ (▪)(1d). C, linear free energy analysis plotting the Taft parameter (σ^*) of the N-fluoroacetyl substituent of 3F4NP-GlcNAc substrate analogues (1a-1d) against the second-order rate constant log V_max/[E]_o K_m values measured for each substrate with SpGH85.

FIGURE 3.

The structure of SpGH85. A, divergent stereo schematic representation of the 1.4 Å crystal structure of the catalytic region of SpGH85. The N-terminal catalytic domain (yellow) is followed by the D1 domain (purple) and then the D2 domain (blue). Relevant active site residues are shown in green stick representation. B, divergent stereo surface representation of SpGH85 shown from the same perspective as in A. The bound NAG-thiazoline molecule from the NAG-thiazoline complex is shown for reference. The surface areas contributed by relevant active residues are shown in purple (Tyr-373), green (acid/base, Glu-337), and blue (catalytic asparagine, Asn-335). The arrows approximate the parts of the active site that may be occupied by a branched, high mannose substrate.

FIGURE 4.

The structure of SpGH85 in complex with a mechanism-based inhibitor. A, electron density of NAG-thiazoline (blue stick representation) bound in the active site of SpGH85 in divergent stereo. The maximum likelihood (47)/σ_a-weighted (68) 2F_obs - F_calc electron density map for the NAG-thiazoline is shown in magenta mesh and contoured at 1σ (0.37 e^-/ų). The F_obs - F_calc omit map of NAG-thiazoline is shown in green mesh and contoured at 2.5σ (0.2 e^-/ų). B, interactions in the SpGH85 active site. NAG-thiazoline is shown in blue stick representation, and side chains that interact with NAG-thiazoline are shown in gray stick representation and labeled; possible hydrogen bonds are shown as dashed lines. The maximum likelihood/σ_a-weighted 2F_obs - F_calc electron density omit maps F_obs - F_calcTyr-373 are shown in magenta mesh and contoured at 1σ (0.37 e^-/ų). The F_obs - F_calc omit map of only the alternate conformation of Tyr-373 is shown in green mesh and contoured at 2.5σ (0.2 e^-/ų). C, cutaway view of the SpGH85 active site surface (transparent gray solvent-accessible surface) when Tyr-373 is in the conformation that does not permit NAG-thiazoline binding. This conformation is also found in the uncomplexed SpGH85 proteins. NAG-thiazoline is shown in yellow stick representation and Tyr-373 in blue stick representation. D, as in C except with Tyr-373 in its alternative conformation that allows NAG-thiazoline binding.

FIGURE 5.

Orientation of Asn-335. A, modeled orientation of Asn-335 where Nδ2 is in close proximity of the NAG-thiazoline N2. The B-factors for Oδ1 and Nδ2 obtained for an unrestrained refinement of the uncomplexed structure (indicated as apo) are shown in black text. The corresponding values for the NAG-thiazoline complex are shown, although because of the lower resolution of this structure these values were obtained with a standard restrained refinement. Bond lengths for the side chain in the uncomplexed structure are indicated in red. A 2F_obs - F_calc electron density map contoured at 4 σ (1.76 e^-/ų) obtained from unrestrained refinement of the uncomplexed structure with the side chain of Asn-335 omitted is shown in green mesh. The F_obs - F_calc electron density map contoured at 12 σ (0.88 e^-/ų) from the same refinement is shown in red mesh. B, same as in A with Asn-335 refined in the alternate conformation. Electron density maps are the same as those in A.

FIGURE 6.

A structurally conserved proton shuttle and its role in the proposed catalytic mechanism of SpGH85. A, superposition of catalytic residues of SpGH85 and a representative member of GH18 both in complex with ligands. The structure of SpGH85 in complex with NAG-thiazoline, in blue, is superimposed over the structure of the S. marcescens ChiB GH18 in complex with allosamidin, in orange (for simplicity only the allosamizoline residue is shown, PDB code 1E6R). The number of catalytic amino acids is shown in the appropriate color for each complex. B, superposition of catalytic residues of SpGH85 and a representative member of GH56 both in complex with ligands. The structure of SpGH85 in complex with NAG-thiazoline, in blue, is superimposed over the structure of the bee venom hyaluronidase GH56 (PDB code 2FCV) in complex with hyaluronic acid tetrasaccharide, in orange (for simplicity only the terminal sugar-like allosamizoline residue is shown). C, schematic showing the proposed catalytic mechanism of SpGH85 with the putative proton shuttle composed of the catalytic residues indicated. In this mechanism Asn-335 acts as the imidic acid tautomer with the nitrogen oriented to accept a hydrogen bond from the substrate amide. Asn-335 acts as a general base to facilitate formation of an oxazoline intermediate, and the proton shuttle acts to disperse charge within the active site. D, schematic showing an alternate catalytic mechanism of SpGH85. In this mechanism Asn-335 is the amide tautomer with the oxygen oriented to accept a hydrogen bond from the substrate amide. Catalysis is driven by cleavage of the glycosidic bond with Glu-337 acting as the general acid and Asn-335 acts to orient the acetamido group and offer some stabilization though hydrogen bonding interactions with the substrate amide.