Dataset Information

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

ABSTRACT: Endo-N-acetyl-?-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-?-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 (?/?)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 ?-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-?-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.

SUBMITTER: Stals I

PROVIDER: S-EPMC3408457 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Stals Ingeborg I Karkehabadi Saeid S Kim Steve S Ward Michael M Van Landschoot Anita A Devreese Bart B Sandgren Mats M

PloS one 20120730 7

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 t ...[more]

PMID: 22859955

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Project description:BackgroundThe ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) is one of the most prolific producers of biomass-degrading enzymes and frequently termed an industrial workhorse. To compete for nutrients in its habitat despite its shortcoming in certain degradative enzymes, efficient perception and interpretation of environmental signals is indispensable. A better understanding of these signals as well as their transmission machinery can provide sources for improvement of biotechnological processes.ResultsThe genome of H. jecorina was analysed for the presence and composition of common signal transduction pathways including heterotrimeric G-protein cascades, cAMP signaling, mitogen activated protein kinases, two component phosphorelay systems, proteins involved in circadian rhythmicity and light response, calcium signaling and the superfamily of Ras small GTPases. The results of this survey are discussed in the context of current knowledge in order to assess putative functions as well as potential impact of alterations of the respective pathways.ConclusionImportant findings include an additional, bacterial type phospholipase C protein and an additional 6-4 photolyase. Moreover the presence of 4 RGS-(Regulator of G-protein Signaling) proteins and 3 GprK-type G-protein coupled receptors comprising an RGS-domain suggest a more complex posttranslational regulation of G-protein signaling than in other ascomycetes. Also the finding, that H. jecorina, unlike yeast possesses class I phosducins which are involved in phototransduction in mammals warrants further investigation. An alteration in the regulation of circadian rhythmicity may be deduced from the extension of both the class I and II of casein kinases, homologues of which are implicated in phosphorylation of FRQ in Neurospora crassa. On the other hand, a shortage in the number of the pathogenicity related PTH11-type G-protein coupled receptors (GPCRs) as well as a lack of microbial opsins was detected. Considering its efficient enzyme system for breakdown of cellulosic materials, it came as a surprise that H. jecorina does not possess a carbon sensing GPCR.

Dataset Information

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

Publications

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

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