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STD‐NMR‐Based Protein Engineering of the Unique Arylpropionate‐Racemase AMDase G74C |
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| Authors: | Sarah Katharina Gaßmeyer Hiroyuki Yoshikawa Junichi Enoki Nadine Hülsemann Prof Raphael Stoll Prof Dr Kenji Miyamoto Prof Dr Robert Kourist |
| Affiliation: | 1. Junior Research Group for Microbial Biotechnology, Ruhr‐Universit?t Bochum, Universit?tsstrasse 150, 44780 Bochum (Germany);2. Department of Bioscience and Informatics, Keio University, 3‐14‐1 Hiyoshi, Kohoku‐ku, Yokohama 223‐8522 (Japan);3. Department of Biomolecular NMR Spectroscopy, Ruhr‐Universit?t Bochum, Universit?tsstrasse 150, 44780 Bochum (Germany) |
| 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. |
| Keywords: | biocatalysis mutagenesis protein engineering racemase STD‐NMR spectroscopy |