title: 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.
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D013302:Streptomyces / Q000201:enzymology*; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D011503:Transglutaminases; D010450:Endopeptidases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases