SUMMARY
Aryl-aldehydes are a common feature in fungal polyketides, which are considered to be exclusively generated by the R domain of non-reducing polyketide synthases (NR-PKSs). However, by cloning and heterologous expression of two cryptic NR-PKS and non-ribosomal peptide synthase (NRPS)-like genes from Aspergillus terreus in Saccharomyces cerevisiae, we discovered 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 such a mechanism may be widely used throughout the fungi kingdom.
␣-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent ␣32 nAChRselective ␣-conotoxins have been identified, currently characterized ␣-conotoxins show no or only weak affinity for ␣42 nAChRs, which are, besides ␣7 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 ␣4 subunit by the corresponding residues in the ␣3 subunit. Two-electrode voltage clamp analysis of these mutants revealed increased affinity of ␣-conotoxins MII, TxIA, and [A10L]TxIA at the ␣4(R185I)2 receptor. Conversely, ␣-conotoxin potency was reduced at the reverse ␣3(I186R)2 mutant. Replacement of ␣4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents ␣-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high ␣-conotoxin potency to the ␣42 receptor. Molecular dynamics simulations of homology models with docked ␣-conotoxin indicate that these residues control access to the ␣-conotoxin binding site.
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