Rieske Oxygenase-Catalyzed Oxidative Late-Stage Functionalization during Complex Antifungal Polyketide Biosynthesis.

Rieske oxygenases (ROs) from natural product biosynthetic pathways are a poorly studied group of enzymes with significant potential as oxidative functionalization biocatalysts. A study on the ROs JerL, JerP, and AmbP from the biosynthetic pathways of jerangolid A and ambruticin VS-3 is described. Their activity was successfully reconstituted using whole-cell bioconversion systems coexpressing the ROs and their respective natural flavin-dependent reductase (FDR) partners. Feeding authentic biosynthetic intermediates and synthetic surrogates to these strains confirmed the involvement of the ROs in hydroxymethylpyrone and dihydropyran formation and revealed crucial information about the RO's substrate specificity. The pronounced dependence of JerL and JerP on the presence of a methylenolether allowed the precise temporal assignment of RO catalysis to the ultimate steps of jerangolid biosynthesis. JerP and AmbP stand out among the biosynthetic ROs studied so far for their ability to catalyze clean tetrahydropyran desaturation without further functionalizing the formed electron-rich double bonds. This work highlights the remarkable ability of ROs to highly selectively oxidize complex molecular scaffolds.

Observation of a Transient Reaction Intermediate Illuminates the Mechanochemical Cycle of the AAA-ATPase p97.

The human ATPase p97, also known as valosin containing protein or Cdc48, is a highly abundant AAA+ engine that fuels diverse energy-consuming processes in the human cell. p97 represents a potential target for cancer therapy and its malfunction causes a degenerative disease. Here, we monitor the enzymatic activity of p97 in real time via an NMR-based approach that allows us to follow the steps that couple ATP turnover to mechanical work. Our data identify a transient reaction intermediate, the elusive ADP.Pi nucleotide state, which has been postulated for many ATPases but has so far escaped direct detection. In p97, this species is crucial for the regulation of adenosine triphosphate turnover in the first nucleotide-binding domain. We further demonstrate how the enzymatic cycle is detuned by disease-associated gain-of-function mutations. The high-resolution insight obtained into conformational transitions in both protein and nucleotide bridges the gap between static enzyme structures and the dynamics of substrate conversion. Our approach relies on the close integration of solution- and solid-state NMR methods and is generally applicable to shed light on the mechanochemical operating modes of large molecular engines.