Biochemistry

Abstract UFMylation is an important biological process where proteins are post-translationally modified via the covalent attachment of the ubiquitin-like modifier (UBL), ubiquitin-fold modifier-1 (UFM1). UFMylation is analogous to ubiquitination, occurring via the transfer of UFM1 across E1-, E2-, and E3-like enzymes, represented by ubiquitin-like modifier-activating enzyme 5 (UBA5), ubiquitin-fo…

Abstract The oxidative modification of low-density lipoprotein (LDL) is involved in the generation of lipid peroxidation-derived electrophilic aldehydes, which covalently react with apolipoprotein B-100 to form various adducts. Although the oxidative modification of LDL is critical in the pathogenesis of atherosclerosis, the structural properties of adducts in oxidized LDL (oxLDL) remain unclear.…

Abstract Here, we performed biochemical and genetic characterization of a GppA/PPX-family phosphohydrolase from Arabidopsis thaliana (AtGPPA/PPX). Sixteen splice variants of AtGPPA/PPX are deposited in public databases and can be classified into two types based on differences in their N-terminal sequences. Green fluorescent protein fusions with either N-terminus indicated that neither possesses a…

Abstract In this work, we report the discovery of a G-quadruplex DNA-selective and near-infrared (NIR-I) emissive G-quadruplex binder derived from a known styryl-quinolinium-based compound. This molecule is unique in its class, with an emission wavelength above 800 nm, and displays selective G-quadruplex binding. Among the synthesized molecules, compound 2 emerged as a selective and the best G-qu…

Abstract The cleavage of the C10–C4a bond in anthraquinones is a key step in generating ring-opened quinone derivatives in filamentous fungi. GedF, a short-chain dehydrogenase/reductase (SDR) from Aspergillus terreus, together with the dioxygenase GedK, mediates this transformation, yet the enzymatic mechanism of GedF remains unclear. Here, isotope labeling experiments confirm that reduction of Q…

Abstract MMUT is one of only two enzymes in the human proteome that depend on the B12 cofactor for activity. It catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5′-deoxyadenosylcobalamin (AdoCbl) as a radical generator. Clinical mutations in MMUT are inherited in an autosomal recessive manner and are classified as B12-responsive mut– and B12 unresponsive mut0 subgroups. In t…

Abstract Serine, a nonessential amino acid classically defined as a precursor for protein synthesis and one-carbon metabolism, is increasingly recognized as a signaling metabolite that links the cellular metabolic status to regulatory decision-making. Intracellular serine availability is shaped by nutrient conditions, glycolytic flux, and activity of the serine synthesis pathway, and these fluctu…

Abstract Fluorinated succinate analogues were evaluated as mechanistic probes of Mycobacterium tuberculosis isocitrate lyase (MtICL). However, 2,2-difluorosuccinate (1; Ki = 6.1 mM) and 2,2,3-trifluorosuccinate (2; Ki = 23.5 μM) act as reversible noncompetitive inhibitors and meso-2,3-difluorosuccinate (3) displayed slow-onset reversible inhibition (Ki = 30 μM), the 2-fluorosuccinate enantiomers …

Abstract The ubiquitous flavoenzymes typically function as oxidoreductases that comprise several distinct main types, including flavoprotein oxidases (FPOs), flavoprotein dehydrogenases (FPDs), and flavoprotein monooxygenases (FPMOs). FPOs and FPDs catalyze two-electron oxidation reactions of organic substrates, typically dehydrogenations, thereby converting oxidized flavin (Flox) into its fully …

Abstract The interaction between mammalian enabled protein (Mena), an actin regulatory protein, and protein tyrosine phosphatase 1B (PTP1B) is critical for maintaining epidermal growth factor receptor (EGFR) signaling homeostasis and regulating cellular motility. Mena recruits PTP1B to activated EGFR in vivo, facilitating receptor dephosphorylation and limiting invasive signaling. However, overex…

Abstract The association of proteins into amorphous aggregates is linked to various detrimental phenomena, ranging from disease mechanisms to the degradation of biopharmaceuticals. The development of aggregation suppression strategies is of considerable interest. Experiments on aggregation (and aggregation suppression) are commonly performed under accelerated conditions, where proteins are expose…

Abstract Neutral sphingomyelinase 2 (nSMase2) is a membrane-bound enzyme that hydrolyzes sphingomyelin (SM) into ceramide and phosphocholine. By generating the bioactive lipid ceramide, nSMase2 plays a critical role in cell stress responses and in regulating exosomes that package and transfer pathogenic factors, including tau protein and amyloid β. Thus, nSMase2 has been implicated in Alzheimer’s…

Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum–iron protein (MoFeP) and the ATP-dependent reductase iron protein (FeP). Although Mo-nitrogenases are widespread across bacteria and archaea and appear to share conserved mechanistic and structural features, FeP and MoFe…

Tryptophan (Trp) metabolism follows three main branches: the kynurenine (KP), serotonin, and indole (IP) pathways. These pathways generate bioactive metabolites that regulate immune responses, redox balance, neurotransmission, metabolic homeostasis, inflammation, and circadian rhythms. A common theme across these pathways is the activation of the aryl hydrocarbon receptor (AhR). Several metabolit…

Understanding how dioxygen accesses buried catalytic centers in metalloenzymes is critical for elucidating enzymatic kinetics and guiding strategies to modulate catalytic activity. Here, we report over 20 μs of classical molecular dynamics simulations of the PHD2 oxygenase, a metalloenzyme regulating hypoxia signaling via HIF-1α hydroxylation. Our extended simulations reveal multiple dynamic diox…

Adrenal glands are one of the pivotal glands of the human endocrine system. Recently, the extracellular matrix (ECM) of the adrenal capsule and cortex was explored in two fractions: outer fraction (OF) and inner fraction (IF). A significant variation in the ECM proteins' levels, including collagens, was documented. Collagen undergoes a plethora of post-translational modifications (PTMs), exhibiti…

Fidelity of DNA polymerases, defined as their ability to incorporate a correct dNMP opposite an undamaged template nucleotide, is a key factor in ensuring genome stability. Many DNA polymerases have been extensively investigated to understand the structural and biochemical basis of their fidelity. However, the great majority of these studies employed simple primer-template substrates, disregardin…

Optical, microplate reader-based assays are a standard tool for the initial biochemical analysis of SARS-CoV-2 main protease (M^pro) inhibitor candidates. Such assays monitor M^pro-catalyzed substrate proteolysis in order to investigate the kinetic impact of inhibitors under examination on the catalytic reaction. This review outlines the numerous intricate considerations invol…

Microproteins are defined as polypeptides of 100-150 or fewer amino acids. With the integrated application of ribosome profiling (Ribo-Seq), mass spectrometry, and bioinformatic approaches, more microproteins have been identified as being encoded by small open reading frames (sORFs). The majority of microproteins are evolutionarily young and may represent species-specific events. This review high…