Activated transglutaminase from Streptomyces mobaraensis is processed by a tripeptidyl aminopeptidase in the final step

Authors


  • Enzymes: transglutaminase, protein-glutamine:amine γ-glutamyltransferase from Streptomyces mobaraensis (EC 2.3.2.13; SwissProt entry name TGL_STRSS, accession number P81453); TAMEP, transglutaminase activating metalloprotease (SwissProt entry name TAMP_STRMB, accession number P83543); P14, TAMEP inhibitory protein (SwissProt entry name SSIT_STRMB, accession number P83544); trypsin from Bos taurus (EC 3.4.21.4; SwissProt entry name TRY2_BOVIN, accession number Q29463); chymotrypsin from Bos taurus (EC 3.4.21.1; SwissProt entry name CTRA_BOVIN, accession number P00766).

H.-L. Fuchsbauer, Fachbereich Chemie- und Biotechnologie, Fachhochschule Darmstadt, Hochschulstraße 2, D-64289 Darmstadt, Germany. Fax: +49 6151 168641, Tel.: +49 6151 168203, E-mail: fuchsbauer@fh-darmstadt.de

Abstract

Transglutaminase (TGase) from Streptomyces mobaraensis is secreted as a precursor protein which is completely activated by the endoprotease TAMEP, a member of the M4 protease family [Zotzel, J., Keller, P. & Fuchsbauer, H.-L. (2003) Eur. J. Biochem. 270, 3214–3222]. In contrast with the mature enzyme, TAMEP-activated TGase exhibits an additional N-terminal tetrapeptide (Phe-Arg-Ala-Pro) suggesting truncation, at least, by a second protease. We have now isolated from the culture broth of submerged colonies a tripeptidyl aminopeptidase (SM-TAP) that is able to remove the remaining tetrapeptide. The 53-kDa peptidase was purified by ion-exchange and phenyl-Sepharose chromatography and subsequently characterized. Its proteolytic activity was highest against chromophoric tripeptides at pH 7 in the presence of 2 mm CaCl2. EDTA and EGTA (10 mm) both diminished the proteolytic activity by half. Complete inhibition was only achieved with 1 mm phenylmethanesulfonyl fluoride, suggesting that SM-TAP is a serine protease. Alignment of the N-terminal sequence confirmed its close relation to the Streptomyces TAPs. That removal of Phe-Arg-Ala-Pro from TAMEP-activated TGase by SM-TAP occurs in a single step was confirmed by experiments using various TGase fragments and synthetic peptides. SM-TAP was also capable of generating the mature N-terminus by cleavage of RAP-TGase. However, AP-TGase remained unchanged. As SM-TAP activity against chromophoric amino acids such as Pro-pNA or Phe-pNA could not be detected, the tetrapeptide of TAMEP-activated TGase must be removed without formation of an intermediate.

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