Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPI accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications.

The protein cross‐linking enzyme transglutaminase from Streptomyces mobaraensis (MTG) is frequently used to modify therapeutic proteins. In order to reveal the binding mode of glutamine donor substrates, we have now crystallized MTG covalently linked to large inhibitory peptides. A series of peptide structures were examined but DIPIGSKMTG, which was chloroacetylated at serine, was the only inhibitory molecule that resulted in an interpretable density map. We found that, besides the warhead (modified Ser6), Ile4 and Gly5 of the inhibitory peptide occupy the tight but extended hydrophobic bottom of the MTG‐binding cleft. Both termini of the peptide protrude along the cleft walls almost perpendicular to the bottom of the extended cleft. This peptide model suggests a zipper‐like cross‐linking mechanism of self‐assembled substrate proteins by MTG.

Streptomyces mobaraensis is a key player for the industrial production of the protein cross‐linking enzyme microbial transglutaminase (MTG). Extra‐cellular activation of MTG by the transglutaminase‐activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross‐linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N‐terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N‐terminally shortened variants clearly indicated the highly conserved Leu40‐Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40‐Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X‐ray crystallography. While no structural data could be obtained for the N‐terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI‐family proteins. Enzymes Chymotrypsin, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/1.html; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/24/77.html; streptogrisin‐A (SGPA), http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/80.html; streptogrisin‐B (SGPB), http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/81.html; subtilisin BPN’, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/62.html; transglutaminase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/2/13.html; transglutaminase‐activating metalloprotease (TAMP), EC3.4.‐.‐; tri‐/tetrapeptidyl aminopeptidase, EC3.4.11.‐; trypsin, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/4.html. Databases The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).

Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines. Being applied to numerous research fields from material sciences to medicine, mTG enables efficient site‐specific conjugation of molecular architectures that possess suitable recognition motifs. In monoclonal antibodies, the lack of native transamidation sites is bypassed by incorporating specific peptide recognition sequences. Herein, we report a rapid and efficient mTG‐catalyzed bioconjugation that relies on a novel recognition motif derived from its native substrate Streptomyces papain inhibitor (SPIP). Improved reaction kinetics compared to commonly applied sequences were demonstrated for model peptides and for biotinylation of Her2‐targeting antibody trastuzumab variants. Moreover, an antibody–drug conjugate assembled from trastuzumab that was C‐terminally tagged with the novel recognition sequence revealed a higher payload‐antibody ratio than the reference antibody.

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