jump to searchjump to navigationjump to content

Publications - Bioorganic Chemistry

Sort by: Year Type of publication

Displaying results 1 to 2 of 2.

Publications

Böhme, B.; Moritz, B.; Wendler, J.; Hertel, T. C.; Ihling, C.; Brandt, W.; Pietzsch, M.; Enzymatic activity and thermoresistance of improved microbial transglutaminase variants Amino Acids 52, 313-326, (2020) DOI: 10.1007/s00726-019-02764-9

Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein β-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.
Publications

Güttler, B. H.-O.; Cynis, H.; Seifert, F.; Ludwig, H.-H.; Porzel, A.; Schilling, S.; A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues Amino Acids 44, 1205-1214, (2013) DOI: 10.1007/s00726-012-1454-0

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day−1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.
IPB Mainnav Search