Biochemistry Biochemistry最新文献

{"title":"Glycosylation Weakens Skp1 Homodimerization in Toxoplasma gondii by Interrupting a Fuzzy Interaction","authors":"Donovan A. Cantrell,&nbsp;Ramona J. Bieber Urbauer,&nbsp;Hyun W. Kim,&nbsp;Robert J. Woods,&nbsp;Jeffrey L. Urbauer,&nbsp;Zachary A. Wood and Christopher M. West*,&nbsp;","doi":"10.1021/acs.biochem.4c0085910.1021/acs.biochem.4c00859","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00859https://doi.org/10.1021/acs.biochem.4c00859","url":null,"abstract":"<p >Skp1/Cullin-1/F-Box protein (SCF) complexes represent a major class of E3 ubiquitin ligases responsible for proteomic control throughout eukaryotes. Target specificity is mediated by a large set of F-box proteins (FBPs) whose F-box domains interact with Skp1 in a conserved, well-organized fashion. In the social amoeba <i>Dictyostelium</i>, Skp1 is regulated by oxygen-dependent glycosylation which alters Skp1’s FBP interactome and inhibits homodimerization that is mediated in part by an ordered interface which overlaps with that of FBPs. Based on sedimentation velocity experiments, Skp1 from the intracellular pathogen <i>Toxoplasma gondii</i> exhibits a homodimerization <i>K</i>_d comparable to that of a previously measured FBP/Skp1 interaction. Glycosylation of Skp1’s disordered C-terminal region (CTR) distal to the ordered homodimer interface significantly weakens Skp1 homodimerization, an effect reproduced by CTR deletion. Replacement with a randomized CTR sequence retains high affinity excluding an extension of the ordered dimer interface. Substitution by poly serine weakens the homodimer to a degree equal to its deletion, indicating a composition dependent effect. The contribution of the CTR to Skp1 homodimerization is canceled by high salt consistent with an electrostatic mechanism. All-atom molecular dynamics simulations suggest that the CTR promotes homodimerization via charge cluster interactions. Taken together, the data indicate that glycosylation weakens homodimerization by disrupting a C-terminal fuzzy interaction that functions in tandem with the ordered dimer interface, thereby freeing Skp1 for FBP binding. Thus, the CTR contributes to Skp1/Skp1 and Skp1/FBP interactions via independent mechanisms that are each influenced by O₂, indicating multiple constraints on the evolution of its sequence.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 10","pages":"2262–2279 2262–2279"},"PeriodicalIF":2.9,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00859","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144088390","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biochemical Studies of a Cyanobacterial Halogenase Support the Involvement of a Dimetal Cofactor","authors":"Michelle L. Wang,&nbsp;Nathaniel R. Glasser,&nbsp;Mrutyunjay A. Nair,&nbsp;Carsten Krebs*,&nbsp;J. Martin Bollinger Jr.* and Emily P. Balskus*,&nbsp;","doi":"10.1021/acs.biochem.4c0072010.1021/acs.biochem.4c00720","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00720https://doi.org/10.1021/acs.biochem.4c00720","url":null,"abstract":"<p >Halogenation is a prominent transformation in natural product biosynthesis, with over 5000 halogenated natural products known to date. Biosynthetic pathways accomplish the synthetic challenge of selective halogenation, especially at unactivated <i>sp</i>³ carbon centers, using halogenase enzymes. The halogenase CylC, discovered as part of the cylindrocyclophane (<i>cyl</i>) biosynthetic pathway, performs a highly selective chlorination reaction on an unactivated <i>sp</i>³ carbon center and is proposed to use a dimetal cofactor. Putative dimetal halogenases are widely distributed across cyanobacterial biosynthetic pathways. However, rigorous <i>in vitro</i> biochemical and structural characterization of these enzymes has been challenging. Here, we report additional bioinformatic analyses of putative dimetal halogenases and the biochemical characterization of a newly identified CylC homologue. Site-directed mutagenesis identifies highly conserved putative metal-binding residues, and Mössbauer spectroscopy provides direct evidence for the presence of a diiron cofactor in these halogenases. These insights suggest mechanistic parallels between diiron and mononuclear nonheme iron halogenases, with the potential to guide further characterization and engineering of this unique subfamily of metalloenzymes.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 10","pages":"2173–2180 2173–2180"},"PeriodicalIF":2.9,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00720","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144088391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulation of Genome Architecture in Huntington’s Disease","authors":"Stephanie Portillo-Ledesma,&nbsp;Minna Hang and Tamar Schlick*,&nbsp;","doi":"10.1021/acs.biochem.5c0002910.1021/acs.biochem.5c00029","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00029https://doi.org/10.1021/acs.biochem.5c00029","url":null,"abstract":"<p >Huntington’s disease (HD) is a neurological condition caused by an excessive expansion of CAG repeats in the Huntingtin (HTT) gene. Although experiments have shown an altered epigenetic landscape and chromatin architecture upon HD development, the structural consequences on the HTT gene remain elusive. Structural data are only available for model nucleosome systems and yeast systems with human nucleosomes. Here, we use our experimentally validated nucleosome-resolution mesoscale chromatin model to investigate folding changes of the HTT gene associated with HD. We investigate how the histone fold domain of the variant macroH2A1, a biomarker of HD, affects the genome structure by modeling HD-like systems that contain (i) 100% canonical, (ii) 100% macroH2A1, (iii) 50% canonical and 50% macroH2A1, and (iv) 100% hybrid cores (one canonical H2A and one macroH2A1 per nucleosome). Then, we model the mouse HTT gene in healthy and HD conditions by incorporating the CAG expansion and macroH2A1 cores, reducing the linker histone density and tail acetylation levels, and incorporating genomic contacts. Overall, our results show that the histone fold domain of macroH2A1 affects chromatin compaction in a fiber-dependent manner (i.e., nucleosome distribution dependent) and can thus both enhance or repress HTT gene expression. Our modeling of the HTT gene shows that HTT is less compact in the diseased condition, which could accelerate the production of the mutated protein. By suggesting the structural biophysical consequences of the HTT gene under HD conditions, our findings may help in the development of diagnostic and therapeutic treatments for HD.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"2100–2115 2100–2115"},"PeriodicalIF":2.9,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.5c00029","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient Assembly of Cytochrome-Based Protoporphyrin IX Composite and Its Characterization as a Photosensitizer","authors":"Kenta Oshima,&nbsp;Makishi Ohnishi,&nbsp;Sheikh Muhammad Ibrahim,&nbsp;Takanori Nishioka and Hiroshi Nakajima*,&nbsp;","doi":"10.1021/acs.biochem.4c0071810.1021/acs.biochem.4c00718","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00718https://doi.org/10.1021/acs.biochem.4c00718","url":null,"abstract":"<p >Apo-<i>r</i>C₅₅₂ C14A/M69F, a heme-deficient mutant of cytochrome <i>c</i>₅₅₂ from <i>Thermus thermophilus</i> HB8, forms a thermally stable composite with protoporphyrin IX (PPIX). However, the apoprotein yield was compromised because of contamination of the purified protein with the holo-protein bearing a covalently attached heme moiety, impeding efficient composite preparation and subsequent studies on the composite. A newly prepared <i>r</i>C₅₅₂ mutant involving quadruple mutations, C11A, C14A, H15F, and M69F (<i>r</i>C₅₅₂-qm), addressed this problem while preserving the inherent thermal stability of a thermophilic bacterial protein. The results obtained for the R125A mutant of <i>r</i>C₅₅₂-qm corroborated the hypothesis that PPIX occupies the cavity of <i>r</i>C₅₅₂-qm with an orientation similar to that of heme <i>c</i> in the wild-type protein. The PPIX composite with <i>r</i>C₅₅₂-qm (PPIX@<i>r</i>C₅₅₂-qm) exhibited superior singlet oxygen (¹O₂) production activity compared to the PPIX composite with human serum albumin (PPIX@HSA). Stern–Volmer quenching analysis suggested that the enhanced ¹O₂ production of PPIX@<i>r</i>C₅₅₂-qm stems from the facilitated access of O₂ to photoexcited PPIX within PPIX@<i>r</i>C₅₅₂-qm relative to PPIX@HSA. Photoexcitation of PPIX@<i>r</i>C₅₅₂-qm induced the self-oxidation of PPIX in an aqueous medium, yielding a composite containing a particular chlorin derivative (photo-PPIX) as the degradation intermediate. The photo-PPIX@<i>r</i>C₅₅₂-qm composite was stable in the solution and showed ¹O₂ production activity upon exposure to red light because of the characteristic of an absorption band of the chlorin ring. This study proposes the <i>r</i>C₅₅₂-qm mutant as a platform for readily creating a stable cytochrome-based photosensitizer responsive to visible and red light in combination with readily available PPIX.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"2089–2099 2089–2099"},"PeriodicalIF":2.9,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Kinetic Mechanism of the Emergent Anticancer Target, Human ADP-ribosyltransferase 1","authors":"Daniel P. Groom,&nbsp;Amanda Lopacinski,&nbsp;Scott J. Garforth and Vern L. Schramm*,&nbsp;","doi":"10.1021/acs.biochem.5c0010510.1021/acs.biochem.5c00105","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00105https://doi.org/10.1021/acs.biochem.5c00105","url":null,"abstract":"<p >Human ADP-ribosyltransferase 1 (<i>hs</i>ART1, EC: 2.4.2.31) is a membrane-associated GPI-anchored, arginine-specific, mono-ADP-ribosyltransferase. The enzyme resides on the endoplasmic reticulum and extracellular cell surface, where it catalyzes the transfer of ADP-ribose (ADPR) from NAD⁺ to arginine residues of neighboring target proteins, forming free nicotinamide (NAM) and N-linked mono-ADP-ribosylation (MARylation) of the target protein. Arginine-specific MARylation regulates the target’s function and cellular roles. Dysregulation of <i>hs</i>ART1 activity has been shown to permit immune cell evasion in non-small cell lung cancer (NSCLC). Inhibition of <i>hs</i>ART1 decreases tumor efficacy and increases T-cell infiltration. <i>hs</i>ART1 is an emerging checkpoint target in select cancers. We performed the first kinetic characterization of the ADP-ribosyltransferase and NAD⁺ glycohydrolase activities of <i>hs</i>ART1. Without an <span>l</span>-arginine substrate, <i>hs</i>ART1 slowly hydrolyses NAD⁺ into NAM and ADPR through an ordered kinetic mechanism. NAD⁺ binding and hydrolysis are followed by the ordered release of NAM followed by ADPR. The ADP-ribosyltransferase activity of <i>hs</i>ART1 to <span>l</span>-arginine-like small molecule substrates gives over a 100-fold improvement in <i>k</i>_cat/<i>K</i>_m and <i>k</i>_cat relative to NAD⁺ hydrolysis. With ADP-ribose acceptors, <i>hs</i>ART1 proceeds through a partially ordered mechanism, whereby the substrate binding of NAD⁺ and <span>l</span>-arginine-like substrate is random. Chemistry proceeds through a ternary complex, and product release is ordered, with NAM first, followed by the ADP-ribosylated acceptor. <i>hs</i>ART1 is not diffusionally rate-limited on <i>k</i>_cat and only partially limited on <i>k</i>_cat/<i>K</i>_m for <span>l</span>-arginine methyl ester. The detailed description of the kinetic mechanism of <i>hs</i>ART1 can aid in the development of novel and selective inhibitors.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"2077–2088 2077–2088"},"PeriodicalIF":2.9,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rational Design of UvsX Recombinase Variants for Enhanced Performance in Recombinase Polymerase Amplification Applications","authors":"Lin Zhang,&nbsp;Enjie Wang,&nbsp;Lvping Wu,&nbsp;Jiaxing Zhang*,&nbsp;Shengping You*,&nbsp;Rongxin Su and Wei Qi,&nbsp;","doi":"10.1021/acs.biochem.5c0009810.1021/acs.biochem.5c00098","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00098https://doi.org/10.1021/acs.biochem.5c00098","url":null,"abstract":"<p >Homologous recombination is a vital biological process for DNA repair, genomic stability, and genetic diversity, driven by the RecA/Rad51 recombinase family. However, as a T4 bacteriophage recombinase homologous to RecA/Rad51, UvsX has limited <i>in vitro</i> performance during recombinase polymerase amplification (RPA) due to ATP utilization and DNA affinity. In this study, UvsX was rationally engineered to enhance these properties through homology modeling, virtual saturation mutations, and consensus mutation strategies. Targeted mutagenesis produced UvsX variants (E198N, E198R, E198K, and K35G) with a 16 ± 4% to 39 ± 6% improvement in RPA activity, while the double mutant K35G/E198R showed an increase of up to 43 ± 4%. Structural analysis revealed that the K35G/E198R mutation enlarged ATP-binding pockets and increased the positive surface potential of DNA-binding sites, resulting in a 12 ± 4% improvement in ATP utilization and more ADP and less AMP generated, a 10 ± 2% enhancement in DNA interaction compared to the wild-type, and better inhibitor tolerance. These findings establish a foundation for the rational optimization of recombinases in nucleic acid amplification and promote their potential for industrial RPA applications.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"2025–2038 2025–2038"},"PeriodicalIF":2.9,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural Elucidation of the Mechanism for Inhibitor Resistance in the Na+-Translocating NADH-Ubiquinone Oxidoreductase from Vibrio cholerae","authors":"Moe Ishikawa-Fukuda,&nbsp;Jun-ichi Kishikawa,&nbsp;Takahiro Masuya,&nbsp;Takeshi Ito,&nbsp;Nicole L. Butler,&nbsp;Danielle McFee,&nbsp;Takayuki Kato,&nbsp;Blanca Barquera,&nbsp;Hideto Miyoshi and Masatoshi Murai*,&nbsp;","doi":"10.1021/acs.biochem.5c0006910.1021/acs.biochem.5c00069","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00069https://doi.org/10.1021/acs.biochem.5c00069","url":null,"abstract":"<p >Na⁺-translocating NADH-ubiquinone oxidoreductase (Na⁺-NQR) is a unique redox-driven Na⁺-pump. Since this enzyme is exclusively found in prokaryotes, including the human pathogens <i>Vibrio cholerae</i> and <i>Neisseria gonorrhoeae</i>, it is a promising target for highly selective antibiotics. Korormicin A, a natural product, and a specific and potent inhibitor of <i>V. cholerae</i> Na⁺-NQR, may become a lead compound for the relevant drug design. We previously showed that the G141A mutation in the NqrB subunit (NqrB-G141A) confers moderate resistance to korormicin A (about 100-fold). However, the efficiency of photoaffinity labeling of the mutant enzyme by a photoreactive korormicin derivative was the same as in the wild-type enzyme. Because of these apparently conflicting results, the molecular mechanism underlying the korormicin A-resistance remains elusive. In the present study, we determined the cryo-EM structure of the <i>V. cholerae</i> NqrB-G141A mutant in the presence of bound korormicin A, and compared it to the corresponding structure from the wild-type enzyme. The toxophoric moiety of korormicin A binds to the mutant enzyme similarly to how it binds to the wild type. However, the added bulk of the alanine-141 excludes the alkyl side chain from the binding cavity, resulting in a decrease in the binding affinity. In fact, isothermal titration calorimetry revealed that the binding affinity of korormicin to the NqrB-G141A mutant is significantly weaker compared to the wild-type. Altogether, we conclude that the inhibitory potency of korormicin A is weaker in the NqrB-G141A mutant due to the decrease in its binding affinity to the altered binding cavity.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"1963–1972 1963–1972"},"PeriodicalIF":2.9,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907270","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of the High-Order Conformational Changes in Glyceraldehyde-3-phosphate Dehydrogenase Induced by Nicotinamide Adenine Dinucleotide, Adenosine Triphosphate, and Oxidants","authors":"Himari Suzuki,&nbsp;Yuki Nicole Makiyama,&nbsp;Yuta Watanabe,&nbsp;Hideo Akutsu,&nbsp;Michiko Tajiri,&nbsp;Yoko Motoda,&nbsp;Ken-Ichi Akagi,&nbsp;Tsuyoshi Konuma,&nbsp;Satoko Akashi and Takahisa Ikegami*,&nbsp;","doi":"10.1021/acs.biochem.4c0079410.1021/acs.biochem.4c00794","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00794https://doi.org/10.1021/acs.biochem.4c00794","url":null,"abstract":"<p >Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis. Beyond this normal function, GAPDH acts as a moonlighting protein, interacting with nonglycolytic molecules to fulfill additional roles, such as apoptosis induction. However, the three-dimensional (3D) structural details underlying these interactions remain unclear, likely due to their dynamic and transient nature. To address this issue, we investigated the structural properties of human and porcine GAPDH using a combination of biophysical techniques, including nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, gel filtration chromatography, and thermal shift assays, with a particular focus on their 3D structures. Our results revealed that although GAPDH becomes unstable upon nicotinamide adenine dinucleotide (NAD⁺) depletion (<i>apo</i> state), its oligomeric structure as a tetramer remains preserved regardless of temperature. In contrast, the presence of adenosine triphosphate (ATP) promotes dimerization at low temperatures, as previously reported. Furthermore, our NMR data suggest that ATP binding exposes the dimer interface and increases the flexibility of side chains in this region. These findings indicate that GAPDH maintains a stable tetrameric structure in the presence of NAD⁺ but becomes structurally unstable and likely more susceptible to oxidation upon NAD⁺ depletion. Additionally, our analyses showed that partial nitrosylation of GAPDH subunits does not induce significant tertiary structural changes. However, significant structural alterations were observed when all four subunits were nitrosylated, although the possibility remains that residues other than the active site residue, Cys152, may have been oxidized. We propose that NAD⁺ depletion, along with oxidation or nitrosylation─most likely at Cys152─destabilizes the GAPDH conformation, and that subsequent ATP binding promotes dimerization. This subunit dissociation may serve as a structural basis for GAPDH’s interactions with other molecules and its moonlighting functions.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"1916–1932 1916–1932"},"PeriodicalIF":2.9,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Decoding the Complex Functional Landscape of the ykkC Riboswitches","authors":"Kathryn M. Barth,&nbsp;David A. Hiller,&nbsp;Gabriel Belem de Andrade,&nbsp;Kumari Kavita,&nbsp;Chrishan M. Fernando,&nbsp;Ronald R. Breaker and Scott A. Strobel*,&nbsp;","doi":"10.1021/acs.biochem.4c0078710.1021/acs.biochem.4c00787","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00787https://doi.org/10.1021/acs.biochem.4c00787","url":null,"abstract":"<p >The <i>ykkC</i> class is the most diverse riboswitch class to date, recognizing structurally and chemically diverse ligands using only minor changes in sequence and structure. Structural studies have demonstrated how sequence changes correspond to altered specificity; however, they are insufficient to define the requirements for functional riboswitch specificity. Here, we report an extensive mutational analysis of the ppGpp riboswitch to investigate the functional role in transcriptional control for this variant riboswitch. Disruption of the terminator hairpin at a single base pair is sufficient to abolish nearly all function, highlighting the fine-tuning of the terminator hairpin to its corresponding aptamer domain. This fine-tuning has been observed in other riboswitches, suggesting that high levels of tunability may be a common feature of riboswitches. Additionally, mutational analysis shows that the previously reported binding site position, G93, does not necessarily correspond to PRPP-driven function as expected. Phylogenetic analysis of natural riboswitches that contain G93 revealed an additional <i>ykkC</i> subclass that binds to both XMP and GMP. This variant subclass is associated with genes for <i>de novo</i> GMP synthesis. Identification of this variant class provides further evidence for small sequence changes corresponding to altered ligand specificity.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"1983–1995 1983–1995"},"PeriodicalIF":2.9,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protonation State Insights into the Influence of Biocatalytic Function for Acetylcholinesterase Mediated by Neonicotinoids","authors":"Zhi-Cong He,&nbsp;Tao Zhang,&nbsp;Wei Peng* and Fei Ding*,&nbsp;","doi":"10.1021/acs.biochem.5c0002410.1021/acs.biochem.5c00024","DOIUrl":"https://doi.org/10.1021/acs.biochem.5c00024https://doi.org/10.1021/acs.biochem.5c00024","url":null,"abstract":"<p >The catalytic efficiency of acetylcholinesterase (AChE) is likely regulated by the protonation states and conformational adaptations of its catalytic residues. While neonicotinoid insecticides are recognized for impairing AChE function through neurotoxic mechanisms, the precise molecular mechanisms governing this inhibition remain poorly characterized. This investigation elucidates how structural variations among neonicotinoids modulate the protonation equilibria of Glu-202 and His-447 in AChE’s catalytic triad. Comparative analysis reveals that nitro-substituted neonicotinoids (imidacloprid, clothianidin) induce more pronounced protonation state transitions compared to their cyano-containing counterparts (thiacloprid, acetamiprid). Specifically, the strong electron-withdrawing nitro groups facilitate the conversion of Glu-202 from the deprotonation (GLU) to protonation (GLH) state and His-447 from the δ- (HID) to ε-position protonation (HIE) state through enhanced electrostatic interactions. These electronic perturbations trigger structural reorganization within the active site, evidenced by nitro group-directed residue realignment and subsequent H-bond formation. Energy decomposition analysis identifies electrostatic contributions as the primary determinant of binding affinity differences, with nitro-neonicotinoids exhibiting stronger interactions than cyano-neonicotinoids. QM/MM metadynamics reveals that substantial protonation state alterations disrupt AChE’s biocatalytic function, particularly its capacity for acetylcholine hydrolysis. Finally, SH-SY5Y-based cellular assays show that imidacloprid exhibits the strongest inhibitory effect on AChE intracellular activity, while thiacloprid and acetamiprid show weaker inhibitory effects, aligning with the computational predictions. This study provides insights into the protonation-state-induced biocatalytic function for acetylcholinesterase mediated by neonicotinoids, contributing to the assessment of exogenous ligand-induced potential ecological and human health risks.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"64 9","pages":"1996–2009 1996–2009"},"PeriodicalIF":2.9,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143907267","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}