Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Takada, Katsumi; Oyama, Hiroshi; Tsunemi, Masahiko; James, Michael N.G.; Oda, Kōhei

doi:10.1016/j.febslet.2005.04.050

Cited by 30 publications

(37 citation statements)

References 22 publications

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“…Large residues with the exception of tryptophan are preferred in the S1 pocket, whereas the S1Ј subsite preferentially binds smaller amino acids such as Gly, Ser, and Ala (Table 2, part B). These data are comparable with subsite specificity profiling of SGP, which indicates that cleavage is favored between bulky P1 residues (Phe Ͼ Tyr Ͼ His) and small P1Ј residues (Ala Ͼ Gly/ Thr Ͼ Ser) (40).…”

Section: Discussionsupporting

confidence: 73%

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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Section: Discussionsupporting

confidence: 73%

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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“…13 The structure-based proposal of the involvement of a Glu-Gln dyad in the catalytic function is supported by mutational analysis of residues Gln53 and Glu136 in SGP. 14 Mutants of SGP (E136A, Q53A and Q53E) lost both the autoprocessing and the enzymatic activities of the wildtype enzyme. Complementary evidence for a GluGln catalytic dyad in glutamic proteases stems from a recent site-directed mutagenesis study on AGP from A. niger, which established as well that residues Gln133 and Glu219 (Gln53 and Glu136 in SGP) constitute the catalytic dyad in that enzyme.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of Scytalidoglutamic Peptidase with its First Potent Inhibitor Provides Insights into Substrate Specificity and Catalysis

Pillai

Cherney

Hiraga

et al. 2007

Journal of Molecular Biology

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“…Analyses of substrate specificities using FRETS-libraries were performed as described previously [13][14][15]. Vimelysin, vimelysin R215D, and thermolysin were dissolved in buffer A (100 mM Tris-HCl, pH 7.5, containing 10 mM CaCl 2 ) and diluted to concentrations of 20 and 2 lg/ml, respectively.…”

Section: Substrate Specificitymentioning

confidence: 99%

Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

et al. 2005

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Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3 0 position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3 0 subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.

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Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Cited by 30 publications

References 22 publications

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

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

Crystal Structure of Scytalidoglutamic Peptidase with its First Potent Inhibitor Provides Insights into Substrate Specificity and Catalysis

Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

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