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J Biol Chem. 2016 Sep 23;291(39):20417-26. doi: 10.1074/jbc.M116.731109. Epub 2016 Aug 4.

Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

Author information

1
Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64287 Darmstadt, Germany, and the Department of Chemistry and.
2
From the Helmholtz-Centre for Infection Research, Braunschweig, 38124 Germany.
3
Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64287 Darmstadt, Germany, and.
4
Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
5
the Department of Chemistry and.
6
Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, 64287 Darmstadt, Germany, and hans-lothar.fuchsbauer@h-da.de.
7
From the Helmholtz-Centre for Infection Research, Braunschweig, 38124 Germany, andrea.scrima@helmholtz-hzi.de.

Abstract

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

KEYWORDS:

Dispase autolysis inducing protein; Streptomyces mobaraensis; X-ray crystallography; enzyme; glutamine cross-linking sites; microbial transglutaminase; protein chemical modification; protein chemistry; tertiary structure

PMID:
27493205
PMCID:
PMC5034039
DOI:
10.1074/jbc.M116.731109
[Indexed for MEDLINE]
Free PMC Article

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