Inhibition of a secreted glutamic peptidase prevents growth of the fungustalaromyces emersonii

O'Donoghue, Anthony J.
Mahon, Cathal S.
Goetz, David H.
O'Malley, James M.
Gallagher, Denise M.
Zhou, Min
Murray, Patrick G.
Craik, Charles S.
Tuohy, Maria G.
O'Donoghue, Anthony J. Mahon, Cathal S.; Goetz, David H.; O'Malley, James M.; Gallagher, Denise M.; Zhou, Min; Murray, Patrick G.; Craik, Charles S.; Tuohy, Maria G. (2008). Inhibition of a secreted glutamic peptidase prevents growth of the fungustalaromyces emersonii. Journal of Biological Chemistry 283 (43), 29186-29195
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 ligno-colum, 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 x 10(5) M-1 s(-1)). Maximum hydrolysis occurs at pH 3.4 and 50 degrees 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.
American Society for Biochemistry & Molecular Biology (ASBMB)
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