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

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 m m CaCl 2 . EDTA and EGTA (10 m m ) both diminished the proteolytic activity by half. Complete inhibition was only achieved with 1 m m 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.