A novel transglutaminase substrate from Streptomyces mobaraensis triggers autolysis of neutral metalloproteases.

Transglutaminase (TGase) from Streptomyces mobaraensis is a Ca(2+) independent enzyme that cross-links proteins to high molecular weight aggregates. A dispase autolysis inducing protein (DAIP) was identified as an intrinsic TGase substrate exhibiting accessible glutamine and lysine residues. DAIP modification during culture by TGase resulted in deamidation of reactive glutamines, formation of glutamic/lysine residue pairs, and failure of cross-linking. The reactivity of modified DAIP can be restored to some extent by N-lauroylamido-3-N',N'-dimethylpropyl amine, thus exposing concertedly buried glutamines and lysines. The novel TGase substrate differs considerably from the well known Streptomyces subtilisin inhibitors in higher molecular mass (37 kDa), lower pI (7.1-7.2), moderate thermo-stability, and the mode of erasing dispase activity. Our experiments suggested that DAIP induces autolysis without removal of essential metals, such as Ca(2+) and Zn(2+). Among other endoproteases, only thermolysin was similarly affected, but at considerably higher DAIP concentrations, due to simultaneous degradation of DAIP.

Inhibition of bacterial transglutaminase by its heat-treated pro-enzyme.

Transglutaminases form a unique family of cross-linking enzymes which may be interesting for pharmaceutical and technical purposes. Bacterial transglutaminase, differing from the eucaryotic counterparts in being independent from Ca2+ ions, is excreted by several Streptomyces species. Until now an endogenous factor regulating activated transglutaminase could not be detected. Here, we investigated whether an inhibitor of transglutaminase is excreted into the culture fluid of Streptomyces mobaraensis. We could demonstrate that heat-resistant inhibitory activity is produced after 24h of growth reaching a maximum after 72h. A two-step ion exchange chromatography purification procedure revealed co-elution of the heat-treated inhibitor with pro-transglutaminase. Experiments with wild-type and recombinant pro-transglutaminase confirmed that the precursor protein indeed inhibits the activity of the mature enzyme.

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 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.