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.

Highlights

  • Endo-N-acetyl-b-D-glucosaminidases (ENGases, EC.3.2.1.96) hydrolyze the b-1,4 linkage in the chitobiose core of N-linked glycans and are capable of releasing entire glycans from glycoproteins leaving one N-acetylglucosamine residue on the substrate

  • Staining of the proteins present in the media showed cellulases with a higher molecular weight in the Endo T knock-out strain compared to the wild type strain (Fig. 1A, lanes 3 and 4)

  • The presence of the N-glycans was further proven by ESI-MS analysis of the catalytic domain of H. jecorina Cel7A: the core protein originating from the wild type strain has been partially deglycosylated due to ENGase activity in the medium while the cellulase from the knock-out strain still contains its three N-glycans (Fig 1B)

Read more

Summary

Introduction

Endo-N-acetyl-b-D-glucosaminidases (ENGases, EC.3.2.1.96) hydrolyze the b-1,4 linkage in the chitobiose core of N-linked glycans and are capable of releasing entire glycans from glycoproteins leaving one N-acetylglucosamine residue on the substrate. The first biochemically characterized representatives, Endo T from the ascomycete Hypocrea jecorina [6], and Endo FV from the basidiomycete Flammulina velutipes [7], show low sequence homology with the bacterial ENGases and with the fungal chitinases. This deglycosylating activity is widely distributed [7] and several highly homologous proteins or gene products are found among ascomycetes [6]. The H. jecorina endo-N-acetyl-b-D-glucosaminidase, Endo T, corresponds with Chi as described by Karlsson et al in a large phylogenetic study [8] but the enzyme is shown in a previous study not to be involved in chitin degradation [6]

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call