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Mutagenesis of Key Residues in the Binding Center of l‐ Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H)
Author(s) -
Xu Xiaoshu,
Chen Jun,
Wang Qingzhuo,
Duan Chunlan,
Li Yan,
Wang Renxiao,
Yang Sheng
Publication year - 2016
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500534
Subject(s) - cofactor , nad+ kinase , escherichia coli , biochemistry , mutant , dehydrogenase , enzyme , mutagenesis , biology , chemistry , stereochemistry , gene
l ‐Aspartate‐β‐semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate pathway. In bacteria, ASADH is highly specific for the cofactor NADP + rather than NAD + . Limited information on cofactor utilization is available, and neither the wild‐type protein nor the available mutants could utilize NAD + efficiently. In this study, we identified several residues crucial for cofactor utilization by Escherichia coli ASADH ( ec ASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ec ASADH‐Q350N and ec ASADH‐Q350N/H171A, which exhibited markedly improved NAD + utilization, were further investigated by various biochemical approaches and molecular modeling. Relative to the wild type, the two mutants showed approximately 44‐fold and 66‐fold increases, respectively, in the constant k cat / K m of NAD + . As desired, they could also utilize NADH efficiently to synthesize l ‐homoserine in cascade reactions in vitro.