title: Site-directed mutations of the gatekeeping loop region affect the activity of Escherichia coli spermidine synthase.
abstract: Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis.
authors.affiliation: Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan.
authors.forename: Mon-Juan
authors.initials: MJ
authors.lastname: Lee
authors.forename: Ya-Ting
authors.initials: YT
authors.lastname: Yang
authors.forename: Vivian
authors.initials: V
authors.lastname: Lin
authors.forename: Haimei
authors.initials: H
authors.lastname: Huang
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011436:Spermidine Synthase / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011436:Spermidine Synthase; D000409:Alanine