Enzymatic cis-Decalin Formation in Natural Product Biosynthesis.

Stereoselective synthesis of cis-decalin structures using [4 + 2] cycloaddition is challenging. We explored the biosynthetic pathway of the fungal natural product fischerin (1) to identify a new pericyclase FinI that can catalyze such a reaction. The cocrystal structure of FinI, a predicted O-methyltransferase, with the product and SAM provides insight into cis-decalin formation in nature.

Macrocyclic Peptide-Conjugated Tip for Fast and Selective Molecular Recognition Imaging by High-Speed Atomic Force Microscopy.

Fast and selective recognition of molecules at the nanometer scale without labeling is a much desired but still challenging goal to achieve. Here, we show the use of high-speed atomic force microscopy (HS-AFM) for real-time and real-space recognition of unlabeled membrane receptors using tips conjugated with small synthetic macrocyclic peptides. The single-molecule recognition method is validated by experiments on the human hepatocyte growth factor receptor (hMET), which selectively binds to the macrocyclic peptide aMD4. By testing and comparing aMD4 synthesized with linkers of different lengths and rigidities, we maximize the interaction between the functionalized tip and hMET added to both a mica surface and supported lipid bilayers. Phase contrast imaging by HS-AFM enables us to discriminate nonlabeled hMET against the murine MET homologue, which does not bind to aMD4. Moreover, using ligands and linkers of small size, we achieve minimal deterioration of the spatial resolution in simultaneous topographic imaging. The versatility of macrocyclic peptides in detecting unlimited types of membrane receptors with high selectivity and the fast imaging by HS-AFM broaden the range of future applications of this method for molecular recognition without labeling.

An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites.

Iterative Catalysis in the Biosynthesis of Mitochondrial Complex II Inhibitors Harzianopyridone and Atpenin B.

The pentasubstituted pyridine natural products harzianopyridone and atpenins are potent inhibitors of mitochondrial complex II. We identified the pathways of these compounds from their fungal producers and uncovered that the biosynthetic steps require multiple iterative enzymes. In particular, a methyltransferase and a flavin-dependent monooxygenase are used iteratively to introduce C5 and C6 methoxy groups. The pathway unexpectedly requires the installation and removal of an N-methoxy group, which is proposed to be a directing group that tunes the reactivity of the pyridone ring, possibly through the alpha effect.