Trapping of a Polyketide Synthase Module after C-C Bond Formation Reveals Transient Acyl Carrier Domain Interactions.

Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.

Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Molecular Characterization and Genotype-Phenotype Correlation of G6PD Mutations in Five Ethnicities of Northern Vietnam.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder and is caused by G6PD gene mutations. To date, more than 400 variants in the G6PD gene have been discovered, and about 160 identified variants are associated with a significant decrease in the G6PD enzyme activity. However, the molecular characterization and epidemiological study of G6PD deficiency are still limited in Vietnam. Therefore, we conducted this study to determine the G6PD variants among the Vietnamese populations and evaluate their correlation to G6PD enzyme activity. A total of 339 patients (302 males and 37 females) were enrolled in this study. The G6PD variants were identified by Sanger sequencing. Our results indicate that males are more severely deficient in G6PD than females. This enzyme activity in males (1.27 ± 1.06 IU/g·Hb) is significantly lower than in females (2.98 ± 1.57 IU/g·Hb) (p < 0.0001). The enzyme activity of the heterozygous-homozygous females and heterozygous females-hemizygous males was found to be significantly different (p < 0.05), which is interpreted due to random X-inactivation. For G6PD molecular characteristics, Viangchan (c.871G>A), Canton (c.1376G>T) and Kaiping (c.1388G>A) variants were the most dominant, accounting for 24.48%, 17.70%, and 22.42%, respectively, whereas the highest frequency of complex variants was observed in Viangchan/Silent with 20.35%. In terms of G6PD activity, the Union variant presented the lowest mean value (1.03 IU/g·Hb) compared to the other variants (p < 0.05). Computational analysis using Polyphen-2 tool investigated that all variants were relative to G6PD deficiency and separated the levels as benign and damaged. The result will establish effective methods to screen G6PD variants in Vietnam.

Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state.

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

Conversion of racemic alcohols to optically pure amine precursors enabled by catalyst dynamic kinetic resolution: experiment and computation.

An unprecedented base metal catalysed asymmetric synthesis of α-chiral amine precursors from racemic alcohols is reported. This redox-neutral reaction utilises a bench-stable manganese complex and Ellman's sulfinamide as a versatile ammonia surrogate. DFT calculations explain the unusual finding of the highly stereoselective transformation enabled by a catalyst that undergoes an unusual dynamic kinetic resolution.

Sustainable Alkylation of Nitriles with Alcohols by Manganese Catalysis.

A general and chemoselective catalytic alkylation of nitriles using a homogeneous nonprecious manganese catalyst is presented. This alkylation reaction uses naturally abundant alcohols and readily available nitriles as coupling partners. The reaction tolerates a wide range of functional groups and heterocyclic moieties, efficiently providing useful cyanoalkylated products with water as the only side product. Importantly, methanol can be used as a C1 source and the chemoselective C-methylation of nitriles is achieved. The mechanistic investigations support the multiple role of the metal-ligand manganese catalyst, the dehydrogenative activation of the alcohol, α-C-H activation of the nitrile, and hydrogenation of the in-situ-formed unsaturated intermediate.

19F NMR as a versatile tool to study membrane protein structure and dynamics.

To elucidate the structures and dynamics of membrane proteins, highly advanced biophysical methods have been developed that often require significant resources, both for sample preparation and experimental analyses. For very complex systems, such as membrane transporters, ion channels or G-protein coupled receptors (GPCRs), the incorporation of a single reporter at a select site can significantly simplify the observables and the measurement/analysis requirements. Here we present examples using 19F nuclear magnetic resonance (NMR) spectroscopy as a powerful, yet relatively straightforward tool to study (membrane) protein structure, dynamics and ligand interactions. We summarize methods to incorporate 19F labels into proteins and discuss the type of information that can be readily obtained for membrane proteins already from relatively simple NMR spectra with a focus on GPCRs as the membrane protein family most extensively studied by this technique. In the future, these approaches may be of particular interest also for many proteins that undergo complex functional dynamics and/or contain unstructured regions and thus are not amenable to X-ray crystallography or cryo electron microscopy (cryoEM) studies.