在嗜热共生细菌(StDAPDH)的内消旋二氨基庚二酸脱氢酶(meso-DAPDH)的脱氨基和胺化过程中,观察到残基R71显示不同的功能。已经提出H154作为碱性残基,其促进水分子在脱氨基期间攻击内消旋DAP的D-手性碳。受R71现象的启发,在本研究中研究了H154在脱氨基和胺化过程中的作用,目的是增强StDAPDH的胺化活性。单点饱和诱变表明,几乎所有的H154突变体都完全失去了对内消旋DAP的脱氨基活性。然而,一些H154变体对丙酮酸和其他大体积的2-酮酸显示出增强的kcat/Km值,如2-氧代戊酸,4-甲基-2-氧代戊酸,2-酮丁酸,和3-甲基-2-氧代丁酸。当与先前报道的W121L/H227I突变体结合时,具有显着改善的kcat/Km值的三重突变体(2.4-,2.5-,2.5-,和4.0倍)获得了这些2-酮酸。尽管以前尝试过,H154位点的突变没有产生预期的结果.此外,这项研究不仅认识到H154对脱氨和胺化反应的独特影响,同时也为蛋白质工程中进一步的高通量筛选和理解StDAPDH的催化机理提供了指导。
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 kcat/Km 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 kcat/Km 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.