Peripheral positions encode transport specificity in the small multidrug resistance exporters.

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

Fungal-derived brevianamide assembly by a stereoselective semipinacolase.

Fungal bicyclo[2.2.2]diazaoctane indole alkaloids represent an important family of natural products with a wide-spectrum of biological activities. Although biomimetic total syntheses of representative compounds have been reported, the details of their biogenesis, especially the mechanisms for assembly of diastereomerically distinct and enantiomerically antipodal metabolites, have remained largely uncharacterized. Brevianamide A represents a basic form of the sub-family bearing a dioxopiperazine core and a rare 3-spiro-ψ-indoxyl skeleton. Here, we identified the Brevianamide A biosynthetic gene cluster from Penicillium brevicompactum NRRL 864 and elucidated the metabolic pathway. BvnE was revealed to be an essential isomerase/semi-pinacolase that specifies selective production of the natural product. Structural elucidation, molecular modeling, and mutational analysis of BvnE, and quantum chemical calculations provided mechanistic insights into the diastereoselective formation of the 3-spiro-ψ-indoxyl moiety in Brevianamide A. This occurs through a BvnE-controlled semi-pinacol rearrangement and a subsequent spontaneous intramolecular [4+2] hetero-Diels-Alder cycloaddition.