High resolution crystal structure of the endo-N-Acetyl-β-D-glucosaminidase responsible for the deglycosylation of Hypocrea jecorina cellulases

Abstract

Endo-N-acetyl-β-D-glucosaminidases (ENGases) hydrolyze the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. The endo-N-acetyl-β-D-glucosaminidases classified into glycoside hydrolase family 18 are small, bacterial proteins with different substrate specificities. Recently two eukaryotic family 18 deglycosylating enzymes have been identified. Here, the expression, purification and the 1.3Å resolution structure of the ENGase (Endo T) from the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei) are reported. Although the mature protein is C-terminally processed with removal of a 46 amino acid peptide, the protein has a complete (β/α)8 TIM-barrel topology. In the active site, the proton donor (E131) and the residue stabilizing the transition state (D129) in the substrate assisted catalysis mechanism are found in almost identical positions as in the bacterial GH18 ENGases: Endo H, Endo F1, Endo F3, and Endo BT. However, the loops defining the substrate-binding cleft vary greatly from the previously known ENGase structures, and the structures also differ in some of the α-helices forming the barrel. This could reflect the variation in substrate specificity between the five enzymes. This is the first three-dimensional structure of a eukaryotic endo-N-acetyl-β-D-glucosaminidase from glycoside hydrolase family 18. A glycosylation analysis of the cellulases secreted by a Hypocrea jecorina Endo T knock-out strain shows the in vivo function of the protein. A homology search and phylogenetic analysis show that the two known enzymes and their homologues form a large but separate cluster in subgroup B of the fungal chitinases. Therefore the future use of a uniform nomenclature is proposed.

Figure 1. Detection of deglycosylating activity.

Figure 1A; SDS-PAGE analysis showing deglycosylating activity: RNAse B band shift analysis with negative control (lane 1), medium of Endo T knock-out transformant 4 (lane 2), medium of RL-P37 wild type strain (lane 3), medium of Endo T knock-out transformant 10 (lane 4) and positive control with purified Endo T (lane 5). Figure 1B; ESI-MS spectrum of the purified catalytic domain from Cel7A secreted by the wild type (a) and Endo T knock-out strain (b). The catalytic core of this protein carries three N-glycans found at Asn45, Asn270 and Asn 384 , ^. The core protein originating from the wild type strain (a) has been partially deglycosylated due to endoglucosaminidase activity in the medium while the protein from the knock-out strain (b) still contains its three N-glycans.

Figure 2. Cartoon representation of the crystal structure of H. jecorina Endo T, top view (a), and side view (b).

The Endo T structure is rainbow colored according to residue number, starting with blue at the N-terminus and ending with red at the C-terminus. In figure (a) the nomenclature of α-helices 1 to 8 and β-strands 1 to 8 building up the (β/α)₈-TIM barrel is indicated. In figure (b) the octasaccharide found bound in the ligand complex structure of E. meningoseptica Endo F3 (PDB ID 1EOM) has been modeled in the active site of Endo T to indicate its position in the enzyme. Single GlcNAc residues at positions N70 and N240 (due to auto-deglycosylation) are shown in stick format and colored orange. Figure prepared with the program PyMol .

Figure 3. Structure based sequence alignment of the four GH family 18 proteins that possess endo-N-acetyl-β-d-glucosaminidase type activity, and with known three-dimensional structure.

The important active site residues are highlighted with a green background. The secondary structure assignment (boxes), indicated on top of the sequence alignment, is rainbow colored according to the residue number, starting with blue at the N-terminus and ending with red at the C-terminus. The shown aligned sequences are (from the top); Elizabethkingia meningoseptica Endo F3 (PDB ID 1EOM, Uniprot access code P36913), Hypocrea jecorina EndoT (PDB ID 4AC1, Uniprot access code C4RA89); Streptomyces plicatus Endo H (PDB ID 1EDT, Uniprot access code P11797.1); Elizabethkingia meningoseptica Endo F1 (PDB ID 2EBN, Uniprot access code P36911.1), and Bacteroides thetaiotaomicron Endo BT (PDB ID 3POH, Uniprot access code Q8A0N4). The glycosylated Asn in the sequons of H. jecorina EndoT are shaded grey. Yellow shaded amino acids are the C-terminal residues observed in the respective crystal structures.

Figure 4. Electron density of the zinc atom bound in the active site of H. jecorina Endo T structure.

The zinc atom, modeled in dual conformation, is shown in grey spheres and the surrounding water molecules that are involved in the coordination spheres of zinc are shown in red spheres. Two water molecules coordinating the zinc atom in the active site have also been modeled in dual confirmation. The displayed maximum likelihood/_σA weighted 2F_obs−F_calc electron density map, contoured at 1.0 σ level (0.38 e/ų), is shown in greyish-blue. Figure prepared with the program PyMol .

Figure 5. Cartoon representation overlay of the superimposed structures of; (a) H. jecorina Endo T, colored in green, and S. plicatus Endo H (PDB ID 1EDT), colored in blue.

The hairpin loop of Endo T, colored in gold, is shorter than the corresponding loop of Endo H, colored in red; (b) H. jecorina Endo T, colored in green, and E. meningoseptica Endo F1 (PDB ID 2EBN), colored in blue. The hairpin loop of Endo T, colored in gold, is shorter than the corresponding loop of Endo F1, colored in red. The loop at the C-terminal of Endo F1 is also colored in red; (c) H. jecorina Endo T, colored in green, and E. meningoseptica Endo F3 (PDB ID 1EOM), colored in red. The hairpin loop is completely missing in the structure of E. meningoseptica Endo F3; (d) H. jecorina Endo T, colored in green, and B. thetaiotaomicron Endo BT (PDB ID 3POH). Figure prepared with the program PyMol .

Figure 6. Overlay of the superimposed active site residues of; (a) H. jecorina Endo T, and S. plicatus Endo H (PDB ID 1EDT); (b) H. jecorina Endo T and E. meningoseptica Endo F1 (PDB ID 2EBN), (c) H. jecorina Endo T, and E. meningoseptica Endo F3 (PDB ID 1EOM), and (d) H. jecorina Endo T, and B. thetaiotaomicron Endo BT (PDB ID 3POH).

The active site residues of Endo T are depicted in orange and those of Endo F1, Endo H, and Endo F3 in red. Figure prepared with the program PyMol .

Figure 7. Structure model surface representations of (a) the H. jecorina Endo T structure, colored in green and (b) Streptomyces plicatus Endo F1 (PDB ID 2EBN), colored in blue.

The extended β1α1, β6α6 and β7α7 loops of H. jecorina EndoT are colored in gold. The C-terminal peptide in Endo F1 is colored in red. The octasaccharide found bound in the ligand complex structure of E. meningoseptica Endo F3 (PDB ID 1EOM) has been modeled in the active site of Endo T. Figure prepared with the program PyMol .

Figure 8. Phylogenetic tree of GH family 18 ENGases, group B.

The phylogenetic tree is based on an amino acid sequence alignment (CLUSTALX) and was constructed by neighbour joining. Bootstrap values are based on 1000 replications and nodes that have bootstrap support above 70% are indicated with the percentage. The tree is rooted with fungal GH family 18 chitinases belonging to the same subgroup. Previously characterized ENGases are indicated with an asterisk*. Boxes indicate proteins belonging to micro-organisms of the same order as described in the text.