Three‐dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the <i>cel7</i> gene from the filamentous fungus, <i>Talaromyces emersonii</i>

Grassick, Alice; Murray, Patrick G.; Thompson, Roisin; Collins, Catherine; Byrnes, Lucy; Birrane, Gabriel; Higgins, Tim; Tuohy, Maria G.

doi:10.1111/j.1432-1033.2004.04409.x

Cited by 106 publications

(73 citation statements)

References 70 publications

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“…The overall fold is homologous to other GH7 cellobiohydrolases, comprising a globular and equatorially elongated protein, but with several interesting features particularly in the surface loop regions (Fig. 2B) (33,(38)(39)(40)(41). The active site tunnel, formed by four pairs of curved antiparallel β-sheets packed face to face, extends from the −7 binding site at Trp57 to the +2 binding site near Phe362, representing a distance of ∼48 Å between the tunnel entrance and exit.…”

Section: Resultsmentioning

confidence: 85%

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Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

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Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.gribble | carbohydrate degrading enzymes

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Section: Resultsmentioning

confidence: 85%

“…The conserved triad of catalytic residues (EXDXXE; Fig. S4A), Glu233, Asp235, and Glu238 use a two-step, retaining mechanism for glycosidic bond hydrolysis (11,33,(38)(39)(40)(41)(42)(43).…”

Section: Resultsmentioning

confidence: 99%

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Kern

McGeehan

Streeter

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

View full text Add to dashboard Cite

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“…Several mesophilic or moderately thermophilic fungal strains are also known to produce enzymes stable and active at high temperatures. These enzymes are produced from species such as Chaetomium thermophila (Venturi et al, 2002), Talaromyces emersonii (Grassick et al, 2004), Thermoascus aurantiacus (Parry et al, 2002). They may be stable at temperatures around 70 °C for prolonged periods.…”

Section: Enzymes For the Cellulose Liquefaction: Thermophilic Enzymesmentioning

confidence: 99%

Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspectives

Verardi¹,

De²,

Ricca³

et al. 2012

Bioethanol

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“…A digoxigenin-labeled DNA probe (Roche Applied Science) was generated using gene-specific oligonucleotides (5Ј-GTGCT-CATCGGCAGCGGCTAC-3Ј and 5Ј-GGCGTTGGTCTCG-CACAGCTG-3Ј). This probe was used to enrich a T. emersonii genomic library (22) for bacteriophage encoding TGP1, and the full-length gene was sequenced using 5Ј-CGAGTGGATCGT-CGAGGACTTCTCCGAG-3Ј and 5Ј-CTCGGAGAAGTCCT-CGACGATCCACTCG-3Ј oligonucleotides.…”

mentioning

confidence: 99%

Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the Fungus Talaromyces emersonii

O’Donoghue

Mahon

Goetz

et al. 2008

Journal of Biological Chemistry

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The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1 such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k cat /K m ‫؍‬ 2 ؋ 10 5 M ؊1 s ؊1 ). Maximum hydrolysis occurs at pH 3.4 and 50°C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K i ‫؍‬ 1.5 nM), as well as a portion of the propeptide sequence, PT1 (K i ‫؍‬ 32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.As chemoheterotrophs, filamentous fungi secrete a variety of polymer-degrading hydrolases such as peptidases and carbohydrases that degrade organic material in the local environment to provide essential nutrients for the growing hyphae. Many fungi release organic acids to acidify the environment while secreting enzymes that are optimally active under these conditions. Traditionally, acid-acting fungal peptidases were assigned to the aspartic protease family and include aspergillopepsin from various Aspergillus species (1) and penicillopepsin from Penicillium species (2). These enzymes have two active-site aspartic acid residues and are strongly inhibited by pepstatin. Recently, two distinct groups of acid peptidases were identified that are insensitive to pepstatin and lack the catalytic motif observed in pepsintype peptidases. One group, "sedolisins" possess a fold similar to members of the subtilisin family but contain an active site triad of serine (S), glutamic acid (E), and aspartic acid (D) (3). They are sensitive to leupeptin and have homologs in bacteria (4), fungi (5), slime mold (6), and mammals (7). The second group of pepstatin-insensitive acid peptidases share a distinct set of structural, enzymatic, and physicochemical properties, and to date have only been identified in a select group of fungi (8). Members of this novel family possess a catalytic dyad consis...

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Three‐dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii

Cited by 106 publications

References 70 publications

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspectives

Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the Fungus Talaromyces emersonii

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