Human pathogenic and commensal bacteria have evolved the ability to scavenge host‐derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known—here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N‐acetyl‐d‐glucosamine, N‐acetyl‐d‐galactosamine and N‐acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N‐acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate—a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism.

The endocannabinoid (eCB) system is an important part of both the human central nervous system (CNS) and peripheral tissues. It is involved in the regulation of various physiological and neuronal processes and has been associated with various diseases. The eCB system is a complex network composed of receptor molecules, their cannabinoid ligands and enzymes regulating the synthesis, release, uptake and degradation of the signalling molecules. Although the eCB system and the molecular processes of eCB signalling have been studied extensively over the last decades, the involved molecules and underlying signalling mechanisms have not been described in full detail. An example pose the two poorly characterized eCB-degrading enzymes α/β-hydrolase domain protein 6 (ABHD6) and ABHD12, which have been shown to hydrolyze 2-arachidonoyl glycerol-the main eCB in the CNS. We review the current knowledge about the eCB system and the role of ABHD6 and ABHD12 within this important signalling system and associated diseases. Homology modelling and multiple sequence alignments highlight the structural features of the studied enzymes and their similarities, as well as the structural basis of disease-related ABHD12 mutations. However, homologies within the ABHD family are very low, and even the closest homologues have widely varying substrate preferences. Detailed experimental analyses at the molecular level will be necessary in order to understand these important enzymes in full detail.

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Hepatocyte nuclear factor 1A (HNF-1A) is a transcription factor expressed in several embryonic and adult tissues, modulating expression of numerous target genes. Pathogenic variants in the HNF1A gene cause maturity-onset diabetes of the young 3 (MODY3 or HNF1A MODY), characterized by dominant inheritance, age of onset before 25-35 years of age, and pancreatic β-cell dysfunction. A precise diagnosis alters management as insulin can be exchanged with sulfonylurea tablets and genetic counselling differs from polygenic forms of diabetes. More knowledge on mechanisms of HNF-1A function and the level of pathogenicity of the numerous HNF1A variants identified by exome sequencing is required for precise diagnostics. Here, we have structurally and biophysically characterized an HNF-1A protein containing both the DNA binding domain and the dimerization domain. We also present a novel approach to characterize HNF-1A variants. The folding and DNA binding capacity of two established MODY3 HNF-1A variant proteins (P112L, R263C) and one variant of unknown significance (N266S) were determined. All three variants showed reduced functionality compared to the wild-type protein. While the R263C and N266S variants displayed reduced binding to an HNF-1A target promoter, the P112L variant was unstable in vitro and in cells. Our results support and mechanistically explain disease causality for all investigated variants and allow for the dissection of structurally unstable and DNA binding defective variants. This points towards structural and biochemical investigation of HNF-1A being a valuable aid in reliable variant classification needed for precision diagnostics and management.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirmed this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. Data are available via ProteomeXchange with identifier PXD021408.