Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease

Authors


  • Proteins and enzymes: Streptomyces subtilisin inhibitor from Streptoverticillium orinoci (SwissProt entry name SSI_STRON, accession number P80597); Streptomyces subtilisin inhibitor from S. coelicolor (SwissProt entry name SSI3_STRCO, accession number P29608). Transglutaminase, protein-glutamine: amine γ-glutamyltransferase from Streptomyces mobaraensis (EC 2.3.2.13, SwissProt entry name Q8KRJ2, accession number Q8KRJ2); dispase, Bacillus polymyxa neutral proteinase (EC 3.4.24.4, no entry); thermolysin, thermophilic-bacterial proteinase from B. thermoproteolyticus rokko (EC 3.4.24.27, SwissProt entry name THER_BACTH, accession number P00800); Streptomyces griseus metalloproteinase II or neutral proteinase, mycolysin (EC 3.4.24.31, no entry).

H.-L. Fuchsbauer, Fachbereich Chemie- und Biotechnologie, Fachhochschule Darmstadt, Hochschulstraße 2, D-64289 Darmstadt, Germany. Fax: +49 6151168641, Tel.: +49 6151168203, E-mail: fuchsbauer@fh-darmstadt.de

Abstract

Streptomyces mobaraensis secretes a Ca2+-independent transglutaminase (TGase) that is activated by removing an N-terminal peptide from a precursor protein during submerged culture in a complex medium [Pasternack, R., Dorsch, S., Otterbach, J. T., Robenek, I. R., Wolf, S. & Fuchsbauer, H.-L. (1998) Eur. J. Biochem. 257, 570–576]. However, an activating protease could not be identified, probably because of the presence of a 14-kDa protein (P14) belonging to the Streptomyces subtilisin inhibitor family. In contrast, if the microorganism was allowed to grow on a minimal medium, several soluble proteases were extracted, among them the TGase-activating protease (TAMEP). TAMEP was purified by sequential chromatography on DEAE- and Arg-Sepharose and used to determine the cleavage site of TGase. It was clearly shown that the peptide bond between Phe(−4) and Ser(−5) was hydrolyzed, indicating that at least one additional peptidase is necessary to complete TGase processing, even if TAMEP cleavage was sufficient to obtain total activity. Sequence analysis from the N-terminus of TAMEP revealed the close relationship to a zinc endo-protease from S. griseus. The S. griseus protease differs from other members of the M4 protease family, such as thermolysin, in that it may be inhibited by the Streptomyces subtilisin inhibitor. P14 likewise inhibits TAMEP in approximately equimolar concentrations, suggesting its important role in regulating TGase activity.

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