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

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.

A Novel Transglutaminase Substrate fromStreptomyces mobaraensisTriggers Autolysis of Neutral Metalloproteases

“…For protein sequence analysis, tiny contaminations were removed by borate gel electrophoresis, as described elsewhere. [5][6][7] Sequence analysis was performed by Edman degradation (Procise 494 Protein Sequencer, Applied Biosystems, Weiterstadt, Germany).…”

MICROBIAL TRANSGLUTAMINASE OF INDONESIAN Streptomyces sp. : Screening and Production in several media

“…Streptomyces transglutaminase is initially secreted as a pro-transglutaminase that could subsequently be activated by several exogenous proteases. Furthermore, Zotzel et al (2003) found that endogenous protease could also activ ate S. mobaerensis MTGase. According to Zhang et al (2009) differentiation, in which it is secreted and activated during the differentiation event.…”

Molecular Basis of Methanol-Inducible Gene Expression and Its Application in the Methylotrophic YeastCandida boidinii

“…The pro-peptide appears to be cleaved by a specific metalloprotease in the culture medium of S. mobaraensis. 50,51) Heterologous gene expression of microbial TGase has also been reported using Escherichia coli as the host, although difficulties were encountered in getting TGase secreted in an active form. 52) Recently, secretion of active TGase was reported by coexpression of a subtilisin-like protease in a Streptomyces or Corynebacterium expression system.…”