Biochemistry最新文献

Collapsed State Mediates the Low Fidelity of the DNA Polymerase β I260 Mutant 塌缩状态介导了 DNA 聚合酶 β I260 突变体的低保真度

Biochemistry Pub Date : 2024-09-19 DOI: 10.1021/acs.biochem.4c00263

Carel Fijen, Cristian Chavira, Khadijeh Alnajjar, Danielle L. Sawyer, Joann B. Sweasy

{"title":"Collapsed State Mediates the Low Fidelity of the DNA Polymerase β I260 Mutant","authors":"Carel Fijen, Cristian Chavira, Khadijeh Alnajjar, Danielle L. Sawyer, Joann B. Sweasy","doi":"10.1021/acs.biochem.4c00263","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00263","url":null,"abstract":"DNA polymerase β (Pol β) fills single nucleotide gaps during base excision repair. Deficiencies in Pol β can lead to increased mutagenesis and genomic instability in the cell, resulting in cancer. Our laboratory has previously shown that the I260 M somatic mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260 M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics and the nature and rates of conformational changes using steady-state fluorescence and Förster resonance energy transfer (FRET). Our data indicate that the I260 M mutation affects the fingers region of rat Pol β by creating a “collapsed” state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. I260 M is a temperature-sensitive mutator and binds nucleotides tighter than the WT protein, resulting in reduced fidelity compared to the WT. Additionally, we have generated a kinetic model of WT and I260 M using FRET and single turnover data, which demonstrates that I260 M precatalytic conformation changes differ compared to the WT as it is missing a precatalytic noncovalent step. Taken together, these results suggest that the collapsed state of I260 M may decrease its ability for nucleotide discrimination, illustrating the importance of the “fingers closing” conformational change for polymerase fidelity and accurate DNA synthesis.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142263154","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication 一种双位内体使 Vero 细胞不受蓖麻毒素侵袭

Biochemistry Pub Date : 2024-09-19 DOI: 10.1021/acs.biochem.4c00385

Timothy F. Czajka, David J. Vance, Renji Song, Nicholas J. Mantis

引用次数: 0

Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits 通过古菌 RlmCD 类甲基转移酶的显著特征解码其底物选择性

Biochemistry Pub Date : 2024-09-18 DOI: 10.1021/acs.biochem.4c00401

Sayan Saha, Shankar Prasad Kanaujia

{"title":"Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits","authors":"Sayan Saha, Shankar Prasad Kanaujia","doi":"10.1021/acs.biochem.4c00401","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00401","url":null,"abstract":"5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron–sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142263156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Substrate Specificity of a Methyltransferase Involved in the Biosynthesis of the Lantibiotic Cacaoidin 参与蓝藻生物可可碱生物合成的甲基转移酶的底物特异性

Biochemistry Pub Date : 2024-09-13 DOI: 10.1021/acs.biochem.4c00150

Haoqian Liang, Youran Luo, Wilfred A. van der Donk

{"title":"Substrate Specificity of a Methyltransferase Involved in the Biosynthesis of the Lantibiotic Cacaoidin","authors":"Haoqian Liang, Youran Luo, Wilfred A. van der Donk","doi":"10.1021/acs.biochem.4c00150","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00150","url":null,"abstract":"Modification of the N- and C-termini of peptides enhances their stability against degradation by exopeptidases. The biosynthetic pathways of many peptidic natural products feature enzymatic modification of their termini, and these enzymes may represent a valuable pool of biocatalysts. The lantibiotic cacaoidin carries an N,N-dimethylated N-terminal amine group. Its biosynthetic gene cluster encodes the putative methyltransferase Cao4. In this work, we present reconstitution of the activity of the enzyme, which we termed CaoSC following standardized lanthipeptide nomenclature, using a heterologously produced peptide as the model substrate. In vitro methylation of diverse lanthipeptides revealed the substrate requirements of CaoSC. The enzyme accepts peptides of varying lengths and C-terminal sequences but requires dehydroalanine or dehydrobutyrine at the second position. CaoSC-mediated dimethylation of natural lantibiotics resulted in modestly enhanced antimicrobial activity of the lantibiotic haloduracin compared to that of the native compound. Improved activity and/or metabolic stability as a result of methylation illustrates the potential future application of CaoSC in the bioengineering of therapeutic peptides.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142263252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Determination of Initial Rates of Lipopolysaccharide Transport 测定脂多糖运输的初始速率

Biochemistry Pub Date : 2024-09-12 DOI: 10.1021/acs.biochem.4c00379

Matthew Nava, Sebastian J. Rowe, Rebecca J. Taylor, Daniel Kahne, Daniel G. Nocera

{"title":"Determination of Initial Rates of Lipopolysaccharide Transport","authors":"Matthew Nava, Sebastian J. Rowe, Rebecca J. Taylor, Daniel Kahne, Daniel G. Nocera","doi":"10.1021/acs.biochem.4c00379","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00379","url":null,"abstract":"Nonvesicular lipid trafficking pathways are an important process in every domain of life. The mechanisms of these processes are poorly understood in part due to the difficulty in kinetic characterization. One important class of glycolipids, lipopolysaccharides (LPS), are the primary lipidic component of the outer membrane of Gram-negative bacteria. LPS are synthesized in the inner membrane and then trafficked to the cell surface by the lipopolysaccharide transport proteins, LptB2FGCADE. By characterizing the interaction of a fluorescent probe and LPS, we establish a quantitative assay to monitor the flux of LPS between proteoliposomes on the time scale of seconds. We then incorporate photocaged ATP into this system, which allows for light-based control of the initiation of LPS transport. This control allows us to measure the initial rate of LPS transport (3.0 min–1 per LptDE). We also find that the rate of LPS transport by the Lpt complex is independent of the structure of LPS. In contrast, we find the rate of LPS transport is dependent on the proper function of the LptDE complex. Mutants of the outer membrane Lpt components, LptDE, that cause defective LPS assembly in live cells display attenuated transport rates and slower ATP hydrolysis compared to wild type proteins. Analysis of these mutants reveals that the rates of ATP hydrolysis and LPS transport are correlated such that 1.2 ± 0.2 ATP are hydrolyzed for each LPS transported. This correlation suggests a model where the outer membrane components ensure the coupling of ATP hydrolysis and LPS transport by stabilizing a transport-active state of the Lpt bridge.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142185907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Structural and Functional Characterization of a Novel Class A Flavin Monooxygenase from Bacillus niacini 新型 A 类黄素单加氧酶的结构和功能表征

Biochemistry Pub Date : 2024-09-12 DOI: 10.1021/acs.biochem.4c00306

Brian C. Richardson, Zachary R. Turlington, Sofia Vaz Ferreira de Macedo, Sara K. Phillips, Kay Perry, Savannah G. Brancato, Emmalee W. Cooke, Jonathan R. Gwilt, Morgan A. Dasovich, Andrew J. Roering, Francis M. Rossi, Mark J. Snider, Jarrod B. French, Katherine A. Hicks

{"title":"Structural and Functional Characterization of a Novel Class A Flavin Monooxygenase from Bacillus niacini","authors":"Brian C. Richardson, Zachary R. Turlington, Sofia Vaz Ferreira de Macedo, Sara K. Phillips, Kay Perry, Savannah G. Brancato, Emmalee W. Cooke, Jonathan R. Gwilt, Morgan A. Dasovich, Andrew J. Roering, Francis M. Rossi, Mark J. Snider, Jarrod B. French, Katherine A. Hicks","doi":"10.1021/acs.biochem.4c00306","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00306","url":null,"abstract":"A gene cluster responsible for the degradation of nicotinic acid (NA) in Bacillus niacini has recently been identified, and the structures and functions of the resulting enzymes are currently being evaluated to establish pathway intermediates. One of the genes within this cluster encodes a flavin monooxygenase (BnFMO) that is hypothesized to catalyze a hydroxylation reaction. Kinetic analyses of the recombinantly purified BnFMO suggest that this enzyme catalyzes the hydroxylation of 2,6-dihydroxynicotinic acid (2,6-DHNA) or 2,6-dihydroxypyridine (2,6-DHP), which is formed spontaneously by the decarboxylation of 2,6-DHNA. To understand the details of this hydroxylation reaction, we determined the structure of BnFMO using a multimodel approach combining protein X-ray crystallography and cryo-electron microscopy (cryo-EM). A liganded BnFMO cryo-EM structure was obtained in the presence of 2,6-DHP, allowing us to make predictions about potential catalytic residues. The structural data demonstrate that BnFMO is trimeric, which is unusual for Class A flavin monooxygenases. In both the electron density and coulomb potential maps, a region at the trimeric interface was observed that was consistent with and modeled as lipid molecules. High-resolution mass spectral analysis suggests that there is a mixture of phosphatidylethanolamine and phosphatidylglycerol lipids present. Together, these data provide insights into the molecular details of the central hydroxylation reaction unique to the aerobic degradation of NA in Bacillus niacini.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142186084","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

DNA Replication across α-l-(3′-2′)-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η 由人类 DNA 聚合酶 η 介导的跨 α-l-(3′-2′)-三呋喃糖基核苷酸的 DNA 复制

Biochemistry Pub Date : 2024-09-11 DOI: 10.1021/acs.biochem.4c00387

Rachana Tomar, Pratibha P. Ghodke, Amritraj Patra, Elizabeth Smyth, Alexander Pontarelli, William Copp, F. Peter Guengerich, John J. Chaput, Christopher J. Wilds, Michael P. Stone, Martin Egli

{"title":"DNA Replication across α-l-(3′-2′)-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η","authors":"Rachana Tomar, Pratibha P. Ghodke, Amritraj Patra, Elizabeth Smyth, Alexander Pontarelli, William Copp, F. Peter Guengerich, John J. Chaput, Christopher J. Wilds, Michael P. Stone, Martin Egli","doi":"10.1021/acs.biochem.4c00387","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00387","url":null,"abstract":"α-l-(3′-2′)-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3′ → 2′ versus 5′ → 3′, respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3′-OH of the primer’s terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer’s terminal 3′-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142185908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Peptide-Based Strategies: Combating Alzheimer’s Amyloid β Aggregation through Ergonomic Design and Fibril Disruption 基于多肽的策略：通过人体工程学设计和纤维断裂防治阿尔茨海默氏症淀粉样蛋白β聚集

Biochemistry Pub Date : 2024-09-10 DOI: 10.1021/acs.biochem.4c00371

Ranit Pariary, Gourav Shome, Sujan Kalita, Sourav Kalita, Anuradha Roy, Amaravadhi Harikishore, Kuladip Jana, Dulal Senapati, Bhubaneswar Mandal, Atin Kumar Mandal, Anirban Bhunia

{"title":"Peptide-Based Strategies: Combating Alzheimer’s Amyloid β Aggregation through Ergonomic Design and Fibril Disruption","authors":"Ranit Pariary, Gourav Shome, Sujan Kalita, Sourav Kalita, Anuradha Roy, Amaravadhi Harikishore, Kuladip Jana, Dulal Senapati, Bhubaneswar Mandal, Atin Kumar Mandal, Anirban Bhunia","doi":"10.1021/acs.biochem.4c00371","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00371","url":null,"abstract":"Amyloidosis of amyloid-β (Aβ) triggers a cascade of events, leading to oxidative damage and neuronal death. Therefore, inhibiting Aβ amyloidosis or disrupting the matured fibrils is the primary target to combat progressive Alzheimer’s disease (AD) pathogenesis. Here, we undertake optimization strategies to improve the antiamyloid efficiency of our previously reported NF11 (NAVRWSLMRPF) peptide. Among the series of peptides tested, nontoxic and serum-stable peptide 1 or P1 containing an anthranilic acid residue shows immense potential in not only inhibiting the Aβ42 amyloid formation but also disrupting the mature Aβ42 fibrils into nontoxic small molecular weight soluble species. Our studies provide high-resolution characterization of the peptide’s mechanism of action. With a binding affinity within the micromolar range for both the monomer and aggregated Aβ42, this α/β hybrid peptide can efficiently modulate Aβ amyloidosis while facilitating the clearance of toxic aggregates and enforcing protection from apoptosis. Thus, our studies highlight that incorporating a β-amino acid not only imparts protection from proteolytic degradation and improved stability but also functions effectively as a β breaker, redirecting the aggregation kinetics toward off-pathway fibrillation.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"42 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142185909","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Inhibition of Sirtuin Deacylase Activity by Peroxynitrite 亚硝酸过氧化物对 Sirtuin 脱乙酰酶活性的抑制作用

Biochemistry Pub Date : 2024-09-10 DOI: 10.1021/acs.biochem.4c00257

Kelsey Bohl, Sarah L. Wynia-Smith, Rachel A. Jones Lipinski, Brian C. Smith

{"title":"Inhibition of Sirtuin Deacylase Activity by Peroxynitrite","authors":"Kelsey Bohl, Sarah L. Wynia-Smith, Rachel A. Jones Lipinski, Brian C. Smith","doi":"10.1021/acs.biochem.4c00257","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00257","url":null,"abstract":"Sirtuins are a class of enzymes that deacylate protein lysine residues using NAD+ as a cosubstrate. Sirtuin deacylase activity has been historically regarded as protective; loss of sirtuin deacylase activity potentially increases susceptibility to aging-related disease development. However, which factors may inhibit sirtuins during aging or disease is largely unknown. Increased oxidant and inflammatory byproduct production damages cellular proteins. Previously, we and others found that sirtuin deacylase activity is inhibited by the nitric oxide (NO)-derived cysteine post-translational modification S-nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO–) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO– (donated from SIN-1) to post-translationally modify and inhibit SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity. In response to SIN-1 treatment, inhibition of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity correlated with increased tyrosine nitration. Mass spectrometry identified multiple novel tyrosine nitration sites in SIRT1, SIRT3, SIRT5, and SIRT6. As each sirtuin isoform has at least one tyrosine nitration site within the catalytic core, nitration may result in sirtuin inhibition. ONOO– can also react with cysteine residues, resulting in sulfenylation; however, only SIRT1 showed detectable peroxynitrite-mediated cysteine sulfenylation. While SIRT2, SIRT3, SIRT5, and SIRT6 showed no detectable sulfenylation, SIRT6 likely undergoes transient sulfenylation, quickly resolving into an intermolecular disulfide bond. These results suggest that the aging-related oxidant peroxynitrite can post-translationally modify and inhibit sirtuins, contributing to susceptibility to aging-related disease.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142185910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0