RESUMO
Aryl-aldehydes are a common feature in fungal polyketides, which are considered to be exclusively generated by the R domain of nonreducing polyketide synthases (NR-PKSs). However, by cloning and heterologous expression of both cryptic NR-PKS and nonribosomal peptide synthase (NRPS)-like genes from Aspergillus terreus in Saccharomyces cerevisiae, we identified a distinct mechanism for aryl-aldehyde formation in which a NRPS-like protein activates and reduces an aryl-acid produced by the accompanying NR-PKS to an aryl-aldehyde. Bioinformatics study indicates that such a mechanism may be widely used throughout the fungi kingdom.
Assuntos
Aldeídos/metabolismo , Aspergillus/metabolismo , Policetídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Aldeídos/análise , Aldeídos/química , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Família Multigênica , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/química , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
α-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent α3ß2 nAChR-selective α-conotoxins have been identified, currently characterized α-conotoxins show no or only weak affinity for α4ß2 nAChRs, which are, besides α7 receptors, the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand-binding domain of the α4 subunit by the corresponding residues in the α3 subunit. Two-electrode voltage clamp analysis of these mutants revealed increased affinity of α-conotoxins MII, TxIA, and [A10L]TxIA at the α4(R185I)ß2 receptor. Conversely, α-conotoxin potency was reduced at the reverse α3(I186R)ß2 mutant. Replacement of α4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents α-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high α-conotoxin potency to the α4ß2 receptor. Molecular dynamics simulations of homology models with docked α-conotoxin indicate that these residues control access to the α-conotoxin binding site.
