title: A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases.
abstract: Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs.
authors.affiliation: Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil.
authors.forename: Priscila Oliveira de
authors.initials: PO
authors.lastname: Giuseppe
authors.affiliation: Laboratory of Structural Biology and Crystallography, Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.
authors.forename: Marcelo Leite Dos
authors.initials: MLD
authors.lastname: Santos
authors.affiliation: Embrapa Maize and Sorghum, Sete Lagoas, MG, Brazil.
authors.forename: Sylvia Morais de
authors.initials: SM
authors.lastname: Sousa
authors.affiliation: Department of Horticultural Science, University of Florida, Gainesville, FL, USA.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Koch
authors.affiliation: Centro Infantil Boldrini, Campinas, SP, Brazil; Department of Medical Genetics, Faculty of Medical Sciences, University of Campinas, Campinas, SP, Brazil.
authors.forename: José Andrés
authors.initials: JA
authors.lastname: Yunes
authors.affiliation: Laboratory of Structural Biology and Crystallography, Institute of Chemistry, University of Campinas, Campinas, SP, Brazil. Electronic address: aparicio@iqm.unicamp.br.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Aparicio
authors.affiliation: Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil. Electronic address: mario.murakami@lnbio.cnpem.br.
authors.forename: Mario Tyago
authors.initials: MT
authors.lastname: Murakami
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry* / Q000648:ultrastructure*; D000074408:Aldo-Keto Reductases; D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry / Q000648:ultrastructure; D004789:Enzyme Activation; D062105:Molecular Docking Simulation; D009249:NADP / Q000737:chemistry* / Q000648:ultrastructure*; D010940:Plant Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D010940:Plant Proteins; D009249:NADP; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: AKR4C subfamily; Cofactor binding; Crystal structure; Intermediate state; Plant aldose reductase