Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i>
Jing Zhu,
Hai Hou,
Kun Li,
Xiaoguang Xu,
Chunmei Jiang,
Dongyan Shao,
Junling Shi,
Dachuan Yin
Affiliations
Jing Zhu
School of Food Science, Xinyang Agriculture and Forestry University, New 24 Street of Yangshan New District, Xinyang 464000, China
Hai Hou
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, China
Kun Li
School of Food Science, Xinyang Agriculture and Forestry University, New 24 Street of Yangshan New District, Xinyang 464000, China
Xiaoguang Xu
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, China
Chunmei Jiang
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, China
Dongyan Shao
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, China
Junling Shi
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, China
Dachuan Yin
Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, China
To better understand its mechanism of activity towards higher alcohols, we overexpressed and purified new Geotrichum candidum GDH (GcGDH). The purified GcGDH (50.27 kDa) was then crystallized, and the crystal diffracted to a resolution of 2.3 Å using X-ray diffraction. We found that the GcGDH crystal structure belonged to space group P212121 and was comprised of two hexamers organized into an asymmetric unit, with each subunit consisting of 452 amino acid residues. The binding sites between higher alcohols or L-glutamic acid and GcGDH were consistent. The optimal reaction conditions for GcGDH and hexanol were a pH of 4.0 and temperature of 30 °C, and those for GcGDH and monosodium glutamate (MSG) were a pH of 8.0 and temperature of 20 °C. The Km values for hexanol and MSG were found to be 74.78 mM and 0.018 mM, respectively. Mutating GcGDH Lys 113 to either Ala or Gly caused a dramatic reduction in its catalytic efficiency towards both MSG and hexanol, suggesting that Lys 113 is essential to the active site of GcGDH.
