Biochemistry [JOURNAL]
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200 papers
Lymphotoxin Beta Receptor, but Not Its Lymphotoxin Alpha-Containing Ligands, Is Essential for the Development of Experimental Dermatitis.
Gorshkova EA, Drutskaya MS, Nedospasov SA … +1 more , Gubernatorova EO
Biochemistry (Mosc) · 2026 Apr · PMID 42070982 · Publisher ↗
Allergic contact dermatitis (ACD) is a chronic inflammatory skin disorder the development of which is driven by allergen sensitization in peripheral lymphoid organs and local cutaneous inflammation. Lymphotoxin (LT) and...
Allergic contact dermatitis (ACD) is a chronic inflammatory skin disorder the development of which is driven by allergen sensitization in peripheral lymphoid organs and local cutaneous inflammation. Lymphotoxin (LT) and its receptor LTβR are critical for lymphoid organogenesis and immune regulation in barrier tissues, but their role in ACD pathogenesis remains incompletely defined. This study aimed to delineate differential contribution of the LTβR-dependent signaling in oxazolone-induced dermatitis. We examined Lta knockout (Lta KO) mice, which lack both soluble LTα3 and membrane-bound isoforms LTα1β2/LTα2β1, and the Ltbr knockout (Ltbr KO) mice, both of which lack lymph nodes. ACD was induced by repeated oxazolone application to ear skin, with assessment of clinical severity, inflammation-associated gene expression, serum IgE levels, and immune cell composition in blood and spleen. Contrary to previous reports, the Lta KO mice developed dermatitis comparable to the wild-type (WT) mice, with elevated IgE production. In contrast, the Ltbr KO mice were substantially protected from the disease, exhibiting attenuated clinical inflammation, reduced ear swelling, and decreased expression in the lesional skin at the background of a lower proportion of circulating CD4 T cells. These findings indicate that LTβR-dependent signaling is pathogenic in allergic skin inflammation, while LTα-mediated pathways are dispensable, suggesting a potential role for the other LTβR ligand, LIGHT, in ACD pathogenesis. Notably, ACD developed even in the absence of lymph nodes, highlighting the importance of local, skin-resident LTβR-dependent mechanisms in the disease development.
Parameters of Delayed Neuroinflammation Following Focal Ischemic Stroke in the Mouse Cerebral Cortex Depend on Lesion Size at the Beginning of the Subacute Stage.
Alekseev VI, Kislukhina EN, Lizunova NV … +4 more , Surin AM, Lipina TV, Savostyanov KV, Bakaeva ZV
Biochemistry (Mosc) · 2026 Apr · PMID 42070981 · Publisher ↗
Dynamics of glial activity changes in the subacute and chronic stages of ischemic stroke after small focal injuries remains poorly understood due to complexity of the long-term animal monitoring and data interpretation....
Dynamics of glial activity changes in the subacute and chronic stages of ischemic stroke after small focal injuries remains poorly understood due to complexity of the long-term animal monitoring and data interpretation. The aim of this study was to assess relationship between the delayed morphological changes in nervous tissue after experimental stroke and lesion parameters determined at various time points. For this purpose, photothrombotic ischemia of the cerebral cortex was induced in the C57BL/6J-Tg(Thy1-GCaMP6f)GP5.17Dkim/J mice, which express fluorescent calcium sensor protein GCaMP6f in cortical neurons. Lesion (ischemic core) size was determined using wide-field optical imaging (WFOI) through a cranial window via the GCaMP6f fluorescence at 3 min, 1 day, and 7 days post-photothrombosis. On day 19, brain sections were analyzed using Nissl staining and immunohistochemistry for microglial (Iba1) and astrocytic (GFAP) markers. It was found that the signs of neuroinflammation - changes in glial cell morphology and quantity - persist in the perifocal region even 19 days after ischemia induction, despite the small lesion volume. A significant linear relationship between microglial nuclear area and lesion size on day 7 was identified. Conversely, no significant correlation was found between the lesion sizes determined in the hyperacute phase (3 min) and acute phase (1 day) and cellular parameters (cell count, morphometric parameters). This indicates that the lesion formation in the acute phase is dynamic, and only the lesion size after its stabilization influences long-term stroke outcomes. Absence of a correlation between the delayed glial changes and ischemic core size during the hyperacute and acute phases suggests that therapeutic window for interventions modulating glial activity may extend to the later period after stroke, even with small lesion size. The results also allow us to conclude that it is not necessary to make an amendment for the initial lesion size in the studies of delayed neuroglial processes in preclinical models. In turn, the correlation between the lesion size on day 7 and microglial cell nucleus area on day 19 demonstrates that the lesion size at the end of the acute phase may be one of the prognostic factors for effectiveness of the post-stroke therapy.
Functioning of Synaptic Vesicle Pools: Diversity and Organizational Principles.
Gafurova CR, Petrov AM
Biochemistry (Mosc) · 2026 Apr · PMID 42070980 · Publisher ↗
Presynaptic nerve terminals contain a large number of vesicles filled with neurotransmitters, whose release ensures signal transmission from the presynaptic neuron to the postsynaptic cell. Despite their morphological ho...
Presynaptic nerve terminals contain a large number of vesicles filled with neurotransmitters, whose release ensures signal transmission from the presynaptic neuron to the postsynaptic cell. Despite their morphological homogeneity, synaptic vesicles (SVs) are functionally heterogeneous and are organized into distinct groups (pools) that differ in their ability for exocytosis and mobilization, recycling kinetics, and protein composition. In addition to the classic pools - the readily releasable pool (RRP), recycling pool, and reserve pool - other populations have been identified, including spontaneously recycling vesicles, vesicles of resting pool and superpool. Vesicles from different pools engage in different modes of exocytosis and endocytosis, and the extent of interpool mixing varies depending on the synapse type and physiological or pathological conditions. Changes in the organization of SV pools underlie multiple forms of synaptic plasticity. Furthermore, SV cycling is a target of several pharmacological agents, and its disruption plays a significant role in the pathogenesis of neurodegenerative diseases. This article is a systematic review of SV pools, their organizational features in central and peripheral synapses, and implications of changes in the structure of SV pools in synaptic plasticity, action of drugs, and development of neurological disorders.
Plant Innate Immunity: Crosstalk of Signaling Pathways.
Skulachev BI, Atabekova AK, Lezzhov AA … +1 more , Solovyev AG
Biochemistry (Mosc) · 2026 Apr · PMID 42070979 · Publisher ↗
The innate immunity of plants is a dynamic, multilevel system traditionally divided into pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Despite being activated by different types of receptors loc...
The innate immunity of plants is a dynamic, multilevel system traditionally divided into pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Despite being activated by different types of receptors localized in different cell compartments, PTI and ETI are currently considered interdependent components of a single defense system. This view suggests that, due to various positive interactions between these two pathways, the innate immunity of plants is more than the sum of PTI and ETI. Available data indicate that PTI and ETI enhance each other synergistically, increasing the concentration of signaling molecules, such as components of kinase cascades, reactive oxygen species, calcium ions, and phytohormones. This leads to the activation of defense genes, providing a local response to pathogens and the development of systemic plant resistance.
How Medically Important Antimicrobials Bind to the 30S Ribosomal Subunit in a Bacterial Pathogen.
Manjari SR, Mallery C, Banavali NK
Biochemistry · 2026 Apr · PMID 42059878 · Full text
Ribosomes translate the genetic code in mRNA to synthesize proteins in all living organisms. Decoding of mRNA occurs in the small subunit of the ribosome and is mediated by tRNA anticodons. Regions near the decoding cent...
Ribosomes translate the genetic code in mRNA to synthesize proteins in all living organisms. Decoding of mRNA occurs in the small subunit of the ribosome and is mediated by tRNA anticodons. Regions near the decoding center are a target for antibiotics, such as aminoglycosides and tetracyclines, where their presence results in errors in protein synthesis. More than two decades of high-resolution structural studies have shown how such medically important antimicrobials (MIAs) bind to the small subunit of the bacterial ribosome. Here, we comprehensively analyze these previously reported structures to help understand the variability with which MIAs bind to small subunits of bacterial ribosomes. We previously solved the hibernating 70S ribosome structure of the bacterial pathogen (), the causative agent of Lyme disease, but there is no structure of any MIA bound to this ribosome reported. Our structural analysis makes it possible to use inexpensive computational methods to predict the binding of these MIAs to the ribosomal 30S small subunit. For this, we used structural analogy, restrained energy minimization, and single-point binding free energy computations. We find the single-point binding free energy of the MIAs to be very sensitive to small conformational changes in the MIA and its environment. Incorporating this knowledge in structure-guided design could aid in the development of narrow-spectrum MIAs targeting specific bacterial pathogens.
A Comparative Investigation of the Mannose Binding Interface in DC-SIGN and MRC1 Carbohydrate Recognition Domains with All-Atom Molecular Dynamics Simulations.
Geissler S, Sacquin-Mora S
Biochemistry · 2026 May · PMID 42059790 · Publisher ↗
Protein-carbohydrate interactions play a key role in numerous biological processes, including immune response, and glycan-based ligands that can target specific protein receptors on a cell surface represent promising can...
Protein-carbohydrate interactions play a key role in numerous biological processes, including immune response, and glycan-based ligands that can target specific protein receptors on a cell surface represent promising candidates for therapeutics applications. For example, in retinoblastoma, the DC-SIGN mannose receptor is overexpressed on the surface of pathogenic cells and represents an interesting target for mannose-based ligands. At the same time, these ligands should not bind to the MRC1 receptor, which is expressed by adjacent, healthy, retinal pigment epithelial cells and presents a carbohydrate recognition domain (CRD) similar to the one of DC-SIGN. Therefore, the challenge remains to obtain a detailed picture of the recognition process between carbohydrates and proteins, in order to design effective and selective therapeutic compounds. In this work we used classical, all-atom molecular dynamics (MD) simulations to investigate the interaction between several mannose based ligands and the CRDs from DC-SIGN and MRC1. The analysis of the protein-carbohydrate contacts from the resulting trajectories highlights the variability of the mannose binding modes on both CRDs, and shows how the increased affinity of mannose for the MRC1 CRD can be related to a specific mannose binding state that is not accessible in the DC-SIGN CRD.
Biased Signaling as Allosteric Probe Dependence: Everything Old Is New Again.
Kenakin T
Biochemistry · 2026 May · PMID 42054078 · Publisher ↗
This chapter considers biased signaling as a natural function of GPCRs in the form of probe dependence. Thus, any ligand that changes the conformation of the receptor (agonist, antagonist, allosteric modulator) has the p...
This chapter considers biased signaling as a natural function of GPCRs in the form of probe dependence. Thus, any ligand that changes the conformation of the receptor (agonist, antagonist, allosteric modulator) has the potential to change the natural signaling of the receptor through diverse conformational alterations in the receptor structure. Given this, selectivity is discussed in terms of varying intrinsic efficacy and selective stabilization of receptor states with methods to detect and measure these effects. Lastly, the translation of to complex systems will be considered.
Tetrahydromethanopterin as a Two-Carbon Carrier: Formation of -Ethyl- and ,-Ethylene-Tetrahydromethanopterin in .
Laird MG, Telimaa H, Koivisto J … +1 more , Scheller S
Biochemistry · 2026 May · PMID 42053226 · Full text
Tetrahydromethanopterin (HMPT) is the main carbon carrier in methane metabolism, which allows the interconversion of a formyl group into a methyl group. Although typically described as a single carbon (C1) carrier, we id...
Tetrahydromethanopterin (HMPT) is the main carbon carrier in methane metabolism, which allows the interconversion of a formyl group into a methyl group. Although typically described as a single carbon (C1) carrier, we identified and characterized -ethyltetrahydromethanopterin (-ethyl-HMPT) and ,-(1,1-ethylene)tetrahydromethanopterin (,-ethylene-HMPT) in the model methanogen . The chemical competence of HMPT to accept C2 moieties was assayed through the study of the spontaneous formation of ,-ethylene-HMPT by the condensation of acetaldehyde with HMPT under standard conditions. The rate constant for this reaction was determined to be 1.53 ± 0.05 M s, and the equilibrium constant was determined to be (8.8 ± 0.5) × 10 M, which is ca. 35 times higher compared to the analogous reaction with the more common carbon carrier tetrahydrofolate. Biochemical assays with cell lysate suggest that the observed formation of -ethyl-HMPT relies on the enzymatic reduction of ,-ethylene-HMPT. These findings illustrate the chemical competency of HMPT to promote biocatalysis with two-carbon moieties and highlight that such reactions might be compatible with established HMPT-mediated microbial pathways.
Molecular Basis of α-Glycine C-H Activation by a Nonheme Fe(II)/2-Oxoglutarate Dioxygenase.
Chen M, Wanniarachchi TN, Caranto JD … +3 more , Seabra G, Bruner SD, Ding Y
Biochemistry · 2026 May · PMID 42048655 · Full text
Nonheme Fe(II)/2-oxoglutarate (Fe/2-OG) dioxygenases carry out a broad range of oxidative reactions, yet α-hydroxylation of glycyl residues remains exceedingly rare. Here, we demonstrate that the Fe/2-OG enzyme MysH from...
Nonheme Fe(II)/2-oxoglutarate (Fe/2-OG) dioxygenases carry out a broad range of oxidative reactions, yet α-hydroxylation of glycyl residues remains exceedingly rare. Here, we demonstrate that the Fe/2-OG enzyme MysH from performs this unusual chemistry during mycosporine-like amino acid biosynthesis, converting mono- and disubstituted precursors into palythines and revealing unexpected substrate tolerance. Kinetic isotope effects, detection of a transient hydroxylated intermediate, and glyoxylate byproduct formation support an α-hydroxylation-initiated mechanism. High-resolution crystal structures, complemented by molecular docking, molecular dynamics simulations, and site-directed mutagenesis, define an active-site architecture that positions the glycyl substrate in a near-transition-state geometry. Hybrid QM/MM calculations reveal a low-barrier hydrogen-atom-transfer step followed by hydroxyl rebound and implicate a conserved Trp125 in an electron-transfer network that lowers the activation barrier. Together, these findings establish a mechanistic framework for protein-directed α-glycine C-H activation by nonheme iron enzymes and provide a blueprint for engineering Fe/2-OG dioxygenases to expand the chemical diversity of mycosporines and related natural products.
Mechanistic Investigation of the DNA Ligase I Deficiency Variant A624T.
Zalenski N, Savoie DJ, Gaur A … +1 more , Suo Z
Biochemistry · 2026 May · PMID 42047708 · Publisher ↗
Mammalian cells rely on DNA ligase 1 (LIG1) as the key ligase for DNA replication and single-strand break repair. LIG1 syndrome, a primary immunodeficiency, arises from various missense mutations in LIG1, such as R305Q,...
Mammalian cells rely on DNA ligase 1 (LIG1) as the key ligase for DNA replication and single-strand break repair. LIG1 syndrome, a primary immunodeficiency, arises from various missense mutations in LIG1, such as R305Q, R641L, R771W, and the recently identified A624T. The R641L and R771W variants, positioned within the nucleotidyltransferase (NTD) and oligonucleotide-binding domains, respectively, reduce catalytic efficiency by perturbing interdomain interactions and catalysis-related DNA binding, despite preserving overall DNA affinity. The R305Q mutation in the DNA binding domain (DBD) severely impairs LIG1 function by disrupting DBD-DNA interactions and the major steps of the DNA ligation mechanism. However, the molecular basis of dysfunction for the A624T variant (located in the NTD) has remained poorly defined. To elucidate the molecular basis underlying this pathogenic variant, we investigated the effects of A624T on protein stability, DNA-binding affinity, and pre-steady-state ligation kinetics. We found that the A624T mutation causes a 3-fold reduction in the rate-limiting adenylyl transfer step at saturating Mg and a 34-fold reduction at lower Mg concentrations while having insignificant effects on protein thermostability, DNA-binding affinity, or the nick-sealing rate. This biochemical profile resembles that of R641L and R771W but is clearly distinct from the severely impaired R305Q variant. Molecular modeling suggests that the A624T substitution subtly perturbs the active site by displacing the catalytic Mg ion and weakening the R641-D600 salt bridge. Collectively, our results indicate that A624T causes modest alterations to the LIG1 active site and ligation kinetics, providing mechanistic insight into how this variant contributes to LIG1 syndrome.
Antimycobacterial Peptides: From Natural Product Discovery to AI Guided Design.
Biswas D, Medina SH
Biochemistry · 2026 May · PMID 42045115 · Full text
Mycobacterial pathogens remain major global health threats, exacerbated by both rapid acquisition of antibiotic resistance and the formidable drug diffusion barrier presented by the rigid mycomembrane. These challenges h...
Mycobacterial pathogens remain major global health threats, exacerbated by both rapid acquisition of antibiotic resistance and the formidable drug diffusion barrier presented by the rigid mycomembrane. These challenges have renewed interest in antimycobacterial peptides (AMyPs), a diverse class of short amphiphilic sequences capable of rapidly killing both drug-sensitive and drug-resistant mycobacteria. Beyond their intrinsic potency, AMyPs can synergize with existing antibiotics and exhibit markedly slower resistance development relative to conventional small molecules. In this review, we synthesize recent advances spanning natural bioprospecting, mechanism-guided rational design, and chemical optimization strategies that have yielded increasingly potent and selective AMyP candidates. We further highlight the rapid emergence of artificial intelligence-driven discovery platforms, which leverage machine-learning models trained on curated, mycobacteria-specific data sets to predict and refine novel AMyPs with growing accuracy. Together, these technologic, biologic, and computational advances outline a rapidly expanding landscape for AMyP-based therapeutic development and establish a foundation for next-generation antimycobacterial drug design.
Stimuli-Chromic Oxazolidine Derivatives as Highly Efficient Molecular Probes for Monitoring Fibrillation Kinetics and Intracellular Detection of Amyloid Fibrils.
Tondkar F, Rezaeifar F, Ghorbani M … +2 more , Abdollahi A, Meratan AA
Biochemistry · 2026 May · PMID 42044386 · Publisher ↗
Misfolding and aggregation of proteins into amyloid fibrils is a main pathological hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Thus, development of probes with the potential to cr...
Misfolding and aggregation of proteins into amyloid fibrils is a main pathological hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Thus, development of probes with the potential to cross biological membranes and detect intracellular aggregates is an area of intense research. In the present study, we have reported the synthesis of two new stimuli-chromic oxazolidine derivatives (OX1 and OX2), with aggregation-induced emission (AIE) characteristics, for monitoring fibrillation kinetics and intracellular detection of amyloid fibrils. Although both probes are nonfluorescent in the presence of monomers and soluble oligomers, their binding to amyloid fibrils is accompanied by considerable red fluorescence. We suggest that changes in the polarity of the amyloid fibril microenvironment, caused by structural changes and exposure of hydrophobic regions during the fibrillation process, promote the selective binding and aggregation of these compounds on the surface of amyloid fibrils, leading to their considerable fluorescence emission. Cellular experiments indicate that both dyes are membrane-permeable without any significant cytotoxicity and can detect intracellular fibrils of α-synuclein and human insulin. Molecular docking studies suggest stronger binding affinity of OX2 than that of ThT for monomers and amyloid fibrils. In summary, we believe that properties such as intracellular detection of amyloid fibrils, red fluorescence without any significant interference with autofluorescence, and solid-state solvatochromic and AIE characteristics may distinguish OX1/OX2 from ThT and previously reported probes, making these compounds more suitable candidates for the detection of amyloid aggregates both and .
Multiscale Modeling of the Bacterial Ribosome to Identify Potential Peptide Modulators and Their Allosteric Effects.
Yuce M, Sungur FA, Kurkcuoglu O
Biochemistry · 2026 Apr · PMID 42037256 · Publisher ↗
The bacterial ribosome is a key antibiotic target, yet peptide-based modulation from its functional and allosteric sites is underexplored. We developed a computational pipeline combining SiteMap-derived binding-site dete...
The bacterial ribosome is a key antibiotic target, yet peptide-based modulation from its functional and allosteric sites is underexplored. We developed a computational pipeline combining SiteMap-derived binding-site detection, consensus docking with Glide and rDock, all-atom truncated molecular dynamics (MD), and coarse-grained MD (CGMD) simulations to identify peptide candidates against fourribosomal sites: the decoding center, peptidyl transferase center, a putative binding pocket on 30S, and the intersubunit bridge B8. Consensus-selected peptides recapitulated hallmark contacts of the native inhibitors viomycin and dalfopristin, and their interaction fingerprints delineate site-specific scaffolds that enable prioritization of inhibitor candidates with enhanced ribosomal affinity, thereby guiding the rational design of novel and effective peptide-based therapeutics. Notably, the peptide CycPeptMPDB_2508 exhibited binding affinity across all investigated sites, nominating it as a versatile lead core for antimicrobial peptide design. Dynamic cross-correlation matrices derived from CGMD simulations captured coupled motions between distal regions of the ribosome, while residue interaction network analysis identified hub residues enriched near the putative binding pocket and B8 bridge, outlining putative allosteric pathways linking local pockets to global motions relevant to decoding and domain closure. This work provides a concise, testable framework for ribosome-targeted peptide discovery and, to the best of our knowledge, constitutes the first ribosome-peptide virtual screening study to employ the viparr module for truncated ribosome-peptide complexes, suggesting the potential applicability of this approach to complex systems and broadening its scope.
Understanding Lignin Oxidation by a Two-Domain Multicopper Oxidase from .
Rese M, van Erven G, Kabel MA … +3 more , Stepnov AA, Eijsink VGH, Tuveng TR
Biochemistry · 2026 May · PMID 42037052 · Full text
Bacterial two-domain multicopper oxidases (2dMCOs) represent a structurally distinct class of trimeric multicopper oxidases. Differing considerably from well-characterized monomeric three-domain laccases, type B 2dMCOs h...
Bacterial two-domain multicopper oxidases (2dMCOs) represent a structurally distinct class of trimeric multicopper oxidases. Differing considerably from well-characterized monomeric three-domain laccases, type B 2dMCOs have a T1 active site positioned in a tunnel in the trimer center. The biochemical properties and roles in lignin conversion of 2dMCOs remain poorly understood. Here, we present a comprehensive biochemical characterization of a type B 2dMCO from (MCO) and discuss links between its structural organization and activity. The T1 copper of MCO had a redox potential of 537 mV, but the turnover number (0.4 s) was ∼1000-fold lower than high-turnover fungal laccases. Stopped-flow UV-vis spectroscopy indicated that this low turnover likely reflects slow reoxidation of the enzyme by O, which has not been previously reported for laccases. Despite the steric constraints imposed by its trimeric structure, MCO oxidized the lignin model compound guaiacylglycerol-β-guaiacyl ether, resulting in both oxidative coupling and bond cleavage, and MCO was able to act on oligomeric birch organosolv lignin, promoting net oxidative polymerization. Interestingly, 2,6-dimethoxyphenol oxidation kinetics and the product profile for guaiacylglycerol-β-guaiacyl ether oxidation by MCO were influenced by pH, buffer composition, and ionic strength, suggesting a potential strategy for tailoring product profiles. Together, these findings demonstrated that MCO functions as a laccase and oxidizes phenolic lignin moieties, but its slow rates and trimeric architecture indicate that it is unlikely to efficiently degrade lignin polymers . This study expands the current understanding of bacterial laccase diversity and provides a foundation for exploring other physiological roles of type B 2dMCOs beyond lignin degradation.
Structural Principles of Covalent Flavin Modification in Oxidoreductases.
Tanner JJ
Biochemistry · 2026 May · PMID 42036955 · Full text
Flavin-dependent oxidoreductases participate in a remarkably diverse array of biochemical transformations, enabled by the redox and covalent versatility of the isoalloxazine cofactor. The N5 and C4a atoms of the flavin a...
Flavin-dependent oxidoreductases participate in a remarkably diverse array of biochemical transformations, enabled by the redox and covalent versatility of the isoalloxazine cofactor. The N5 and C4a atoms of the flavin are central to catalysis and, in selected cases, serve as loci of irreversible covalent modification. This review focuses on structurally validated examples of covalent inactivation of flavoenzymes in which the chemical nature of the flavin adduct has been established by X-ray crystallography, beginning with the historically important reversible N5 sulfite adduct and extending to mechanism-based irreversible inactivation. Mining of the Protein Data Bank reveals that covalent flavin modification occurs across multiple enzyme families, including monoamine oxidases, lysine-specific demethylase 1, proline dehydrogenase, spermine/polyamine oxidases, and cytokinin oxidase/dehydrogenase. These systems illustrate a limited but recurring set of structural outcomes, most commonly N5 alkylation or C4a alkylation, frequently accompanied by flavin reduction and characteristic butterfly bending of the isoalloxazine ring. A theme emerging from the structural record is the susceptibility of amine oxidases and dehydrogenases among covalently inactivated flavoenzymes. This prevalence reflects intrinsic mechanistic features of C-N bond oxidation─particularly iminium formation proximal to reduced flavin─that predispose these enzymes to irreversible flavin modification. In contrast, other flavoenzyme classes rarely generate electrophiles positioned for flavin attack, rendering stable covalent modification less common. By integrating structural, mechanistic, and inhibitor-design perspectives, this review highlights both the chemical disposition underlying covalent flavin inactivation and the constraints that shape its distribution across flavoprotein biochemistry.
Structure, Biosynthesis, and Bioactivity of Nostolysamides.
Weir E, Anterola I, van der Donk WA
Biochemistry · 2026 May · PMID 42032808 · Full text
A recent genome-mining study identified class II lanthipeptides encoded in PCC 73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were no...
A recent genome-mining study identified class II lanthipeptides encoded in PCC 73102 that contain acyl groups conjugated to Lys side chains. The structure and bioactivity of these peptides, named nostolysamides, were not determined. In this study, we produced the nostolysamides by coexpression of the NpuA precursor peptide with an N-terminal SUMO tag with the class II lanthipeptide synthetase NpuM in . All four lanthionine and methyllanthionine residues were shown to have the DL configuration by Marfey's analysis. Tandem mass spectrometry and mutagenesis studies indicate an N-terminal nonoverlapping methyllanthionine ring and three overlapping rings at the C-terminus, for which the most likely ring pattern is proposed. The NpuM lanthipeptide synthetase is a member of the ProcM-clade and catalyzes ring formation with both C-to-N and N-to-C directionality. After removal of the leader peptide, the resulting lanthipeptide exhibits antibacterial as well as antifungal activity against species by disrupting cell membranes. Antibacterial activity is shown not to involve lipid II. The biosynthetic gene cluster also encodes an acetyltransferase NpuN that transfers long-chain acyl groups to the side chain of a Lys residue at position 1 of the precursor peptide. studies of NpuN show relatively broad substrate specificity, with NpuN conjugating various acyl groups from acyl-CoA substrates to Lys1 in the nostolysamides. The acylation did not appreciably change the antifungal and antimicrobial activity of nostolysamide, showing that it is not required for these activities.
Cell Signaling by Tryptophan Catabolism.
Torrelli-Diljohn A, Kulkarni B, Vitturi DA
Biochemistry · 2026 May · PMID 42029070 · Full text
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, neurotransmi...
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 metabolites from KP, IP, and serotonin act as endogenous AhR ligands or interact indirectly with AhR, but the downstream consequences of this interaction depend on the cellular environment and inflammatory context. In addition, Trp metabolites also impact other signaling pathways, including GPR35, NMDA receptors, serotonergic receptors, and NAD biosynthesis. Notably, our group recently discovered that the upregulation of the KP results in the formation of the novel redox-active mediator kynurenine-carboxyketoalkene. This finding expands the signaling repertoire of Trp metabolism to include the modulation of cysteine-dependent pathways, with important implications for the maintenance of cellular homeostasis and immune control. Overall, flux through the oxidative arm of the KP links inflammation to cellular energy metabolism, while microbial indole derivatives influence host-mucosal immunity and host-microbe communication. The serotonin pathway connects neuroendocrine signaling with the peripheral nervous system's regulation of metabolism. Shifts in Trp homeostasis caused by inflammation, alterations in microbial composition, or metabolic demand modify downstream signaling outputs under physiological and pathological conditions. In this regard, dysregulation of Trp metabolism is implicated in neurodegeneration, cancer, metabolic disease, cardiovascular dysfunction, and chronic inflammation. This review presents Trp catabolism as a distributed signaling network and offers new insights into its physiological functions.
Mechanistic Insights into Dioxygen Transport Routes in the PHD2 Oxygenase from Long-Time Scale Simulations.
Wiley B, Furini S, Domene C
Biochemistry · 2026 May · PMID 42023757 · Full text
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...
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 dioxygen transport routes from solvent-exposed regions through the cupin fold to the metal active site, capturing transient interconverting channels and kinetic heterogeneity inaccessible to prior short-time scale studies. Dioxygen transport occurs on widely differing time scales, from rapid exchange (∼250 ps) to long residence times within internal hydrophobic cavities lasting hundreds of nanoseconds. These internal cavities act as dynamic reservoirs, modulating dioxygen availability and potentially contributing to the high K and slow oxidative turnover by PHD2. Analysis of cavity-lining residues identifies hydrophobic positions that may be targeted to tune catalytic rates. Collectively, our results refine the mechanistic model of dioxygen access in PHD2 and demonstrate how high-resolution simulations can uncover functionally relevant kinetic landscapes, providing principles applicable to the design and regulation of molecular catalysts.
Gibson Assembly of Highly Repetitive DNA.
Curtician G, Batchik A, Holland NB … +1 more , Turk EM
Biochemistry · 2026 May · PMID 42021475 · Publisher ↗
Highly repetitive DNA sequences remain difficult to synthesize, assemble, and verify, limiting their use in synthetic biology. Here, we report a modular plasmid framework for the scalable, sequence-defined assembly of re...
Highly repetitive DNA sequences remain difficult to synthesize, assemble, and verify, limiting their use in synthetic biology. Here, we report a modular plasmid framework for the scalable, sequence-defined assembly of repetitive DNA. As a model system, plasmids encoding repeats of the pentapeptide Gly-Val-Gly-Val-Pro (GVGVP) were constructed to produce elastin-like polypeptides (ELPs) with temperature-dependent solubility. A synthetic DNA fragment encoding GVGVP was incorporated into a plasmid architecture that enables iterative repeat amplification through a Gibson-based digest-and-assemble workflow. Sequential dIII and HI digestion followed by Gibson Assembly increased repeat number (2n-1 per cycle) while preserving plasmid architecture, yielding constructs up to GVGVP, as verified by whole-plasmid sequencing. A superfolder GFP was added to the GVGVP library with expression of up to 513 repeats and functional characterization up to 257 repeats in NEB 5-alpha cells. These results establish a generalizable strategy for constructing large repetitive DNA sequences and encoding programmable protein polymers.
Profiling Selectivity for the Virulence Factor OspF.
McCurtin NP, Gazaferi A, Novick ND … +3 more , Kim K, Lesser CF, Scheck RA
Biochemistry · 2026 May · PMID 42018782 · Full text
Phosphothreonine lyases are a small family of bacterial virulence factors that are secreted into host cells during infection by some Gram-negative pathogens. Phosphothreonine lyases catalyze an irreversible phosphate β-e...
Phosphothreonine lyases are a small family of bacterial virulence factors that are secreted into host cells during infection by some Gram-negative pathogens. Phosphothreonine lyases catalyze an irreversible phosphate β-elimination that converts phosphothreonine (pThr) to dehydrobutyrine (Dhb) on host proteins. Here, we explore the substrate profile for OspF, a phosphothreonine lyase secreted during infection, which has been previously reported to modify mitogen-activated protein kinases (MAPKs) to silence the host immune response. In this work, we use a combination of assays with synthetic phosphopeptides and bottom-up chemoproteomic profiling to uncover the OspF substrates. These studies not only show that OspF exhibits selectivity within the MAPK family but also reveal that OspF modifies a wide range of cellular targets in cellular lysates and during infection. Using a nucleophilic phosphine chemical probe, we identified and validated new OspF targets beyond the MAPK family, including Rab1A and casein kinase 2β. Together, these findings provide valuable insights about OspF substrates in infection and highlight the need for further studies to reveal its full role in the context of pathogenesis. Moreover, these data underscore the potential utility of OspF and other phospholyases as novel chemical and synthetic biology tools for site-specific protein editing.
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