Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i>
To better understand its mechanism of activity towards higher alcohols, we overexpressed and purified new <i>Geotrichum candidum</i> GDH (<i>Gc</i>GDH). The purified <i>Gc</i>GDH (50.27 kDa) was then crystallized, and the crystal diffracted to a resolution of 2.3...
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2023-06-01
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| Online Access: | https://www.mdpi.com/2073-4352/13/6/980 |
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| author | Jing Zhu Hai Hou Kun Li Xiaoguang Xu Chunmei Jiang Dongyan Shao Junling Shi Dachuan Yin |
| author_facet | Jing Zhu Hai Hou Kun Li Xiaoguang Xu Chunmei Jiang Dongyan Shao Junling Shi Dachuan Yin |
| author_sort | Jing Zhu |
| collection | DOAJ |
| description | To better understand its mechanism of activity towards higher alcohols, we overexpressed and purified new <i>Geotrichum candidum</i> GDH (<i>Gc</i>GDH). The purified <i>Gc</i>GDH (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 <i>Gc</i>GDH 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 <i>Gc</i>GDH were consistent. The optimal reaction conditions for <i>Gc</i>GDH and hexanol were a pH of 4.0 and temperature of 30 °C, and those for <i>Gc</i>GDH and monosodium glutamate (MSG) were a pH of 8.0 and temperature of 20 °C. The <i>K</i>m values for hexanol and MSG were found to be 74.78 mM and 0.018 mM, respectively. Mutating <i>Gc</i>GDH 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 <i>Gc</i>GDH. |
| first_indexed | 2024-03-11T02:36:06Z |
| format | Article |
| id | doaj.art-bb87d10d95264f198d9085de2c227335 |
| institution | Directory Open Access Journal |
| issn | 2073-4352 |
| language | English |
| last_indexed | 2024-03-11T02:36:06Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Crystals |
| spelling | doaj.art-bb87d10d95264f198d9085de2c2273352023-11-18T09:57:26ZengMDPI AGCrystals2073-43522023-06-0113698010.3390/cryst13060980Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i>Jing Zhu0Hai Hou1Kun Li2Xiaoguang Xu3Chunmei Jiang4Dongyan Shao5Junling Shi6Dachuan Yin7School of Food Science, Xinyang Agriculture and Forestry University, New 24 Street of Yangshan New District, Xinyang 464000, ChinaKey Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, ChinaSchool of Food Science, Xinyang Agriculture and Forestry University, New 24 Street of Yangshan New District, Xinyang 464000, ChinaKey Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, ChinaKey Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, ChinaKey Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, ChinaKey Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, ChinaKey Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, ChinaTo better understand its mechanism of activity towards higher alcohols, we overexpressed and purified new <i>Geotrichum candidum</i> GDH (<i>Gc</i>GDH). The purified <i>Gc</i>GDH (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 <i>Gc</i>GDH 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 <i>Gc</i>GDH were consistent. The optimal reaction conditions for <i>Gc</i>GDH and hexanol were a pH of 4.0 and temperature of 30 °C, and those for <i>Gc</i>GDH and monosodium glutamate (MSG) were a pH of 8.0 and temperature of 20 °C. The <i>K</i>m values for hexanol and MSG were found to be 74.78 mM and 0.018 mM, respectively. Mutating <i>Gc</i>GDH 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 <i>Gc</i>GDH.https://www.mdpi.com/2073-4352/13/6/980<i>Geotrichum candidum</i>glutamate dehydrogenasegene cloningcharacterizationcrystal structure |
| spellingShingle | Jing Zhu Hai Hou Kun Li Xiaoguang Xu Chunmei Jiang Dongyan Shao Junling Shi Dachuan Yin Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i> Crystals <i>Geotrichum candidum</i> glutamate dehydrogenase gene cloning characterization crystal structure |
| title | Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i> |
| title_full | Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i> |
| title_fullStr | Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i> |
| title_full_unstemmed | Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i> |
| title_short | Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from <i>Geotrichum candidum</i> |
| title_sort | crystal structure analysis and characterization of nadp dependent glutamate dehydrogenase with alcohols activity from i geotrichum candidum i |
| topic | <i>Geotrichum candidum</i> glutamate dehydrogenase gene cloning characterization crystal structure |
| url | https://www.mdpi.com/2073-4352/13/6/980 |
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