Clp-family proteins are prototypes for studying the mechanism of ATP-dependent proteases because the proteolytic activity of the ClpP core is tightly regulated by activating Clp-ATPases. Nonetheless, the proteolytic activation mechanism has remained elusive because of the lack of a complex structure. Acyldepsipeptides (ADEPs), a recently discovered class of antibiotics, activate and disregulate ClpP. Here we have elucidated the structural changes underlying the ClpP activation process by ADEPs. We present the structures of Bacillus subtilis ClpP alone and in complex with ADEP1 and ADEP2. The structures show the closed-to-open-gate transition of the ClpP N-terminal segments upon activation as well as conformational changes restricted to the upper portion of ClpP. The direction of the conformational movement and the hydrophobic clustering that stabilizes the closed structure are markedly different from those of other ATP-dependent proteases, providing unprecedented insights into the activation of ClpP.

Oxygen-containing heterocycles are a common structural motif in polyketide natural products and contribute significantly to their biological activity. Here, we report structural and mechanistic investigations on AmbDH3, a polyketide synthase domain with dual activity as dehydratase (DH) and pyran-forming cyclase in ambruticin biosynthesis. AmbDH3 is similar to monofunctional DH domains, using H51 and D215 for dehydration. V173 was confirmed as a diagnostic residue for cyclization activity by a mutational study and enzymatic in vitro experiments. Similar motifs were observed in the seemingly monofunctional AmbDH2, which also shows an unexpected cyclase activity. Our results pave the way for mining of hidden cyclases in biosynthetic pathways. They also open interesting prospects for the generation of novel biocatalysts for chemoenzymatic synthesis and pyran-polyketides by combinatorial biosynthesis.

Background: A eukaryotic HslV (TbHslV) protease and two potential HslU (TbHslU1 and TbHslU2) ATPases have been isolated from Trypanosoma brucei. Results: We determined the crystal structure of TbHslV at 2.4 Å resolution. Only TbHslU2 activated TbHslV protease activity. Conclusion: A key tyrosine residue in TbHslU2 required for activating TbHslV was identified. Significance: This study lays the groundwork for understanding the eukaryotic HslVU system.

Biosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular complexity. Enzymes that form complex structural elements with multiple stereocenters, like chiral saturated oxygen heterocycles (CSOH), are of particular interest for a synthetic application, as their use promises to significantly simplify access to these elements. Here, the biocatalytic characterization of AmbDH3, an enzyme that catalyzes intramolecular oxa-Michael addition (IMOMA) is reported. This reaction essentially gives access to various types of CSOH with adjacent stereocenters, but it is not yet part of the repertoire of preparative biocatalysis. An in-depth study on the synthetic utility of AmbDH3 was performed, which made extensive use of complex synthetic precursor surrogates. The enzyme exhibited stability and broad substrate tolerance in in vitro experiments, which was in agreement with the results of molecular modeling. Its selectivity profile enabled kinetic resolution of chiral tetrahydropyrans (THPs) under control of up to four stereocenters. A systematic optimization of the reaction conditions enabled gram-scale conversions yielding preparative amounts of chiral THP. The synthetic utility of AmbDH3 was finally demonstrated by its successful application in the key step of a chemoenzymatic total synthesis to the THP-containing phenylheptanoid (−)-centrolobine. These results highlight the synthetic potential of AmbDH3 and related IMOMA cyclases as a biocatalytic alternative that further develops the available chemical-synthetic IMOMA methodology.