9IHS

Microbial transglutaminase mutant - D3C/G283C

Summary for 9IHS

• Entry DOI: 10.2210/pdb9ihs/pdb
• Descriptor: Protein-glutamine gamma-glutamyltransferase, 2-(N-MORPHOLINO)-ETHANESULFONIC ACID, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: transglutaminase, thermostable mutant, disulfide bond, transferase
• Biological source: Streptomyces mobaraensis
• Total number of polymer chains: 4
• Total formula weight: 152839.13

Authors

Suzuki, M.,Date, M.,Kashiwagi, T.,Takahashi, K.,Nakamura, A.,Tanokura, M.,Suzuki, E.,Yokoyama, K. (deposition date: 2024-06-18, release date: 2024-09-18, Last modification date: 2024-10-16)

Primary citation

Suzuki, M.,Date, M.,Kashiwagi, T.,Takahashi, K.,Nakamura, A.,Tanokura, M.,Suzuki, E.,Yokoyama, K.
Random mutagenesis and disulfide bond formation improved thermostability in microbial transglutaminase.
Appl.Microbiol.Biotechnol., 108:478-478, 2024

PubMed Abstract: Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (k/K) at 37.0 °C was increased from 3.3 × 10 M s for the wild type to 5.9 × 10 M s. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency. KEY POINTS: • Improved heat resistance is essential to broaden the application of MTG. • The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability. • X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.

PubMed: 39354113
DOI: 10.1007/s00253-024-13304-1

Experimental method

X-RAY DIFFRACTION (2 Å)