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Characterization of Ecm14, a Fungal Pseudopeptidase
Author(s) -
Idowu Temitope A,
Schott Matthew J,
Lyons Peter J
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.596.7
Subject(s) - biochemistry , amino acid , saccharomyces cerevisiae , enzyme , active site , gene , carboxypeptidase , histidine , biology , yeast , function (biology) , trypsin , exopeptidase , chemistry , genetics
Many peptidase families contain inactive enzyme homologs due to the alteration of critical active site amino acids. The function of these inactive homologs is in many cases an open question, and their study made more difficult by their lack of activity. For example, the M14 family of metallocarboxypeptidases is a large family of peptidases made up of 23 members in humans; three of these are inactive enzyme homologs for which we have little functional information. Interestingly, the yeast Saccharomyces cerevisiae has only one M14 metallocarboxypeptidase homolog, Ecm14, and this sole homolog is predicted to be inactive. Our goal was to investigate the molecular and biological function of this pseudopeptidase. An analysis of available fungal amino acid sequences showed that the Ecm14 gene was present and conserved in ascomycete fungi only. In order to characterize the function of Ecm14, we expressed a C‐terminally histidine tagged version of Ecm14 using the Sf9/baculovirus system. The protein was secreted and subsequently purified by metal affinity chromatography. Incubation of Ecm14 with trypsin or chymotrypsin resulted in the specific removal of the Ecm14 prodomain. The mature form of Ecm14 lacked any detectable enzymatic activity, but was able to prevent the cleavage of standard substrates by an active metallocarboxypeptidase. Preliminary results from a screen of knockout fitness in response to chemical stressors suggest a biological function for Ecm14. We propose that Ecm14 may perform a regulatory function through binding to specific C‐terminal amino acids, thus blocking their cleavage by active carboxypeptidases. Support or Funding Information Supported by Andrews University Faculty Research Grants to PJL