1. Enhancing the amination activity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by modifying the crucial residue His154 for deamination.
- Author
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Yuan, Kehao, Huo, Zongchao, Zhang, Ya'ning, Guo, Zuran, Chang, Yucan, Jin, Yunming, Gao, Lining, Zhang, Tong, Li, Yanwei, Ma, Qinyuan, and Gao, Xiuzhen
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PYRUVIC acid , *HIGH throughput screening (Drug development) , *AMINATION , *MUTAGENESIS , *ACIDS - Abstract
During the deamination and amination processes of meso -diaminopimelate dehydrogenase (meso -DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso -DAP during deamination. Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso -DAP. However, some H154 variants showed enhanced k cat / K m values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved k cat / K m values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH. • H154 was first experimentally identified to be the fatal residue for deamination. • H154 mutation boosted the amination towards pyruvic acid and bulky 2-keto acids. • Coupling H154 variants with W121L/H227I resulted in enhanced amination activities. • The first report on the contrary effects of H154 during deamination and amination. • A reminder to update the high-throughput screening for DAPDH amination activity. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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