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ABSTRACT Polyketide synthases (PKSs) are multi-domain enzymatic assembly lines that biosynthesise a wide selection of bioactive natural products from simple building blocks. In contrast to theircis-acyltransferase (AT) counterparts,trans-AT PKSs rely on stand-alone AT domains to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery.Trans-AT PKS gene clusters also encode acyl hydrolase (AH) domains, which are predicted to share the overall fold of AT domains, but hydrolyse aberrant acyl chains from ACP domains, thus ensuring efficient polyketide biosynthesis. How such domains specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH domain and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AH and ACP domains contributes to recognition of cognate and non-cognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the production of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis bytrans-AT PKSs, and provide foundations for future bioengineering efforts. 

Small molecule guests influence the functional properties of supramolecular hydrogels. Molecular-level understanding of such sol-gel compositions and structures is challenging due to the lack of long-range order and inherently heterogeneous sol-gel interface. In this study, insight into the uptake process of biologically relevant small molecules into guanosine-quartet(G4) borate hydrogels is obtained by gel-state magic-angle spinning (MAS) NMR spectroscopy. G4∙K + borate hydrogel can absorb up to 0.3 equivalent of cationic methylene blue (MB) without a significant disruption of the G4 fibrils that make up the gel, whereas the addition of over 0.3 equivalents of MB to the same gel leads to a gel-to-sol transition. The gel-to-sol transition process is characterized ex situ by analyzing and comparing the 1 H and 11 B MAS NMR spectra acquired before and after the MB uptake. In particular, 11 B isotropic chemical shifts and quadrupole interactions were determined by analyzing the 11 B MAS NMR spectra acquired at different magnetic fields, 11.7 T, 14.1 T and 20 T, which enable the different local bonding environments of borate anions in sol- and gel domains to be distinguished and identified. By comparison, uptake of heterocyclic molecules such as adenine, cytosine and 1-methylthymine into G4∙Na + borate hydrogels lead to stiff and clear gels while increasing the solubility of the nucleobases as compared to the solubility of the same compounds in water. G4∙Na + gel can uptake one equiv. of adenine with minimal disruption to the sol-gel framework, thus enhancing the adenine solubility up to an order of magnitude as compared to water. Combined multinuclear ( 1 H, 11 B and 23 Na) NMR spectroscopy analysis and vial inversion tests revealed that the nucleobases are embedded into pores of the sol phase rather than being closely interacting with the G-4 fibrils that make up the gel phase. These results indicate that G-4 hydrogels have potential applications as carrier systems for small molecules. Gel-state MAS NMR spectroscopy can be used to gain insight into host-guest interactions in complex heterogeneous sol-gel systems, which is often difficult to obtain from the conventional techniques such as X-ray scattering, electron microscopy and optical spectroscopy.