title: Mutagenesis of Key Residues in the Binding Center of l-Aspartate-b-Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H).
abstract: 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 (ecASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ecASADH-Q350N and ecASADH-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 kcat /Km of NAD(+). As desired, they could also utilize NADH efficiently to synthesize l-homoserine in cascade reactions in vitro.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China.
authors.forename: Xiaoshu
authors.initials: X
authors.lastname: Xu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China.
authors.forename: Qingzhuo
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China.
authors.forename: Chunlan
authors.initials: C
authors.lastname: Duan
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.
authors.forename: Renxiao
authors.initials: R
authors.lastname: Wang
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China. syang@sibs.ac.cn.|Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 30 Meilong Road, Shanghai, 200237, China. syang@sibs.ac.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009243:NAD / Q000378:metabolism*
chemical_list: D009243:NAD; D001223:Aspartate-Semialdehyde Dehydrogenase
keywords: dehydrogenases; enzyme catalysis; iterative saturation mutagenesis; molecular modeling; oxidoreductases