Mara Boenitz-Dulat
Hoffmann-La Roche
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Publication
Featured researches published by Mara Boenitz-Dulat.
Journal of Biological Chemistry | 2017
Wojtek Steffen; Fu Chong Ko; Jigar Patel; Victor Lyamichev; Thomas J. Albert; Jörg Benz; Markus G. Rudolph; Frank Bergmann; Thomas Streidl; Peter Kratzsch; Mara Boenitz-Dulat; Tobias Oelschlaegel; Michael Schraeml
Microbial transglutaminases (MTGs) catalyze the formation of Gln–Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallest MTG characterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis, a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates.
PLOS ONE | 2018
Ilke Ugur; Martin Schatte; Antoine Marion; Manuel Glaser; Mara Boenitz-Dulat; Iris Antes
The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.6 μs) and with bound Ca2+ (simulations of 1.6 μs, 1.8 μs, and 2.5 μs). Our results show that Ca2+ binding causes not only ordering of the disordered β6/β7 loop of the protein, but also modulates hinge motions in the dynamic β7/β8 loop, which is important for the catalytic activity of the enzyme. Cation binding triggers signal transmission from the Ca2+ binding site to the dynamic β7/β8 loop via the repetitive folding/unfolding of short helical stretches of the disordered β6/β7 loop. These correlated structural rearrangements lead to several distinct conformational states of the binding groove, which show optimal binding features for the sorting signal motif and feature binding energies up to 20 kcal/mol more favorable than observed for the sortase A without Ca2+. The presented results indicate a highly correlated, conformational selection-based activation mechanism of the enzyme triggered by cation binding. They also demonstrate the importance of the dynamics of intrinsically disordered regions for allosteric regulation.
Archive | 2005
Mara Boenitz-Dulat; Jessica Laggerbauer; Rainer Schmuck; Peter Kratzsch; Wolfgang-Reinhold Knappe
Archive | 2007
Mara Boenitz-Dulat; Daniela Beck; Peter Kratzsch; Rainer Schmuck; Der Eltz Herbert Von
Archive | 2008
Mara Boenitz-Dulat; Daniela Beck; Peter Kratzsch; Rainer Schmuck; Herbert von der Eltz
Archive | 2005
Mara Boenitz-Dulat; Peter Kratzsch; Rainer Schmuck
Archive | 2016
Mara Boenitz-Dulat; Erhard Kopetzki; Peter Kratzsch; Martin Schatte
Archive | 2017
Mara Boenitz-Dulat; Peter Kratzsch; Martin Schatte
Archive | 2017
Daniela Beck; Mara Boenitz-Dulat; Peter Kratzsch; Thomas Streidl; Carina Horn
Archive | 2017
Mara Boenitz-Dulat; Martin Schatte