STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C

Sarah Katharina Gaßmeyer, Hiroyuki Yoshikawa, Junichi Enoki, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

Original languageEnglish
Pages (from-to)1943-1949
JournalChemBioChem
Volume16
Issue number3
DOIs
Publication statusPublished - 23 Jun 2015
Externally publishedYes

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Racemases and Epimerases
Protein Engineering
Nuclear magnetic resonance
Amino Acids
Substrates
Enzymes
Carboxylic Acids
Catalysis
Mutagenesis
Proteins
Magnetic Resonance Spectroscopy
Spectroscopic analysis
Nuclear magnetic resonance spectroscopy
Reaction rates
Substitution reactions
Atoms
Experiments

Fields of Expertise

  • Human- & Biotechnology

Cite this

Gaßmeyer, S. K., Yoshikawa, H., Enoki, J., Hülsemann, N., Stoll, R., Miyamoto, K., & Kourist, R. (2015). STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C. ChemBioChem, 16(3), 1943-1949. https://doi.org/10.1002/cbic.201500253

STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C. / Gaßmeyer, Sarah Katharina; Yoshikawa, Hiroyuki; Enoki, Junichi; Hülsemann, Nadine; Stoll, Raphael; Miyamoto, Kenji; Kourist, Robert.

In: ChemBioChem, Vol. 16, No. 3, 23.06.2015, p. 1943-1949.

Research output: Contribution to journalArticleResearchpeer-review

Gaßmeyer, SK, Yoshikawa, H, Enoki, J, Hülsemann, N, Stoll, R, Miyamoto, K & Kourist, R 2015, 'STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C' ChemBioChem, vol. 16, no. 3, pp. 1943-1949. https://doi.org/10.1002/cbic.201500253
Gaßmeyer SK, Yoshikawa H, Enoki J, Hülsemann N, Stoll R, Miyamoto K et al. STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C. ChemBioChem. 2015 Jun 23;16(3):1943-1949. https://doi.org/10.1002/cbic.201500253
Gaßmeyer, Sarah Katharina ; Yoshikawa, Hiroyuki ; Enoki, Junichi ; Hülsemann, Nadine ; Stoll, Raphael ; Miyamoto, Kenji ; Kourist, Robert. / STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C. In: ChemBioChem. 2015 ; Vol. 16, No. 3. pp. 1943-1949.
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