title: Protein motifs involved in coenzyme interaction and enzymatic efficiency in anabaena ferredoxin-NADP+ reductase.
abstract: Ferredoxin-NADP+ reductases (FNRs) must determine the coenzyme specificity and allow the transient encounter between N5 of its flavin cofactor and C4 of the coenzyme nicotinamide for efficient hydride transfer. Combined site-directed replacements in different putative determinants of the FNR coenzyme specificity were simultaneously produced. The resulting variants were structurally and functionally analyzed for their binding and hydride transfer abilities to the FNR physiological coenzyme NADP+/H, as well as to NAD+/H. The previously studied Y303S mutation is the only one that significantly enhances specificity for NAD+. Combination of mutations from the pyrophosphate or 2'-phosphate regions, even including Y303S, does not improve activity with NAD+, despite structures of these FNRs show how particular coenzyme-binding regions resembled motifs found in NAD+/H-dependent enzymes of the FNR family. Therefore, the "rational approach" did not succeed well, and coenzyme specificity redesign in the FNR family will be more complex than that anticipated in other NADP+/NAD+ families.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, and Institute of Biocomputation and Physics of Complex Systems, Universidad de Zaragoza, 50009 Zaragoza, Spain.
authors.forename: José R
authors.initials: JR
authors.lastname: Peregrina
authors.forename: Beatriz
authors.initials: B
authors.lastname: Herguedas
authors.forename: Juan A
authors.initials: JA
authors.lastname: Hermoso
authors.forename: Marta
authors.initials: M
authors.lastname: Martínez-Júlvez
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
mesh_terms: D017033:Anabaena / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding
chemical_list: D003067:Coenzymes; D006859:Hydrogen; D005287:Ferredoxin-NADP Reductase