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Biochemical characterization and crystal structures of a fungal family 3 ?-glucosidase, Cel3A from Hypocrea jecorina.

ABSTRACT: Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant ?-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the ?-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)-?-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A). Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 ?-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn(208) and Asn(310). In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.

SUBMITTER: Karkehabadi S

PROVIDER: S-EPMC4223358 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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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.

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Biochemical characterization and crystal structures of a fungal family 3 ?-glucosidase, Cel3A from Hypocrea jecorina.

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