Pub Date : 2024-09-19DOI: 10.1021/acs.biochem.4c00263
Carel Fijen, Cristian Chavira, Khadijeh Alnajjar, Danielle L. Sawyer, Joann B. Sweasy
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.
DNA 聚合酶 β（Pol β）可在碱基切除修复过程中填补单核苷酸缺口。Pol β的缺陷会导致细胞内突变和基因组不稳定性增加，从而引发癌症。我们的实验室先前已经证明，首次在前列腺癌中发现的 Pol β I260 M 体细胞突变会以序列上下文依赖的方式降低核苷酸的识别能力。在这种情况下，I260 M 比 WT 更容易将不正确的 G 与 A 相反结合。为了确定 I260M 保真度降低的分子机制，我们利用单次周转动力学研究了掺入情况，并利用稳态荧光和佛斯特共振能量转移（FRET）研究了构象变化的性质和速率。我们的数据表明，I260 M 突变会影响大鼠 Pol β 的手指区，在开放（无核苷酸时）和封闭（化学反应前）状态下产生一种 "塌陷 "状态。I260 M是一种温度敏感突变体，与核苷酸的结合比WT蛋白更紧密，导致保真度比WT蛋白低。此外，我们还利用 FRET 和单次周转数据生成了 WT 和 I260 M 的动力学模型，结果表明 I260 M 的前催化构象变化与 WT 蛋白不同，因为它缺少一个前催化非共价步骤。综上所述，这些结果表明，I260 M 的塌缩状态可能会降低它对核苷酸的辨别能力，这说明了 "手指闭合 "构象变化对聚合酶保真度和准确 DNA 合成的重要性。
{"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}
Pub Date : 2024-09-19DOI: 10.1021/acs.biochem.4c00385
Timothy F. Czajka, David J. Vance, Renji Song, Nicholas J. Mantis
Expression of camelid-derived, single-domain antibodies (VHHs) within the cytoplasm of mammalian cells as “intrabodies” has opened up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein. The intrabody consists of two structurally defined VHHs that target distinct epitopes on RT’s enzymatic subunit (RTA): V9E1 targets RTA’s P-stalk recruitment site, and V2A11 targets RTA’s active site. Resistance to RT conferred by the biparatopic VHH construct far exceeded that of either of the VHHs alone and effectively inhibited all measurable RT-induced cytotoxicity in vitro. We propose that the targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.
{"title":"A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication","authors":"Timothy F. Czajka, David J. Vance, Renji Song, Nicholas J. Mantis","doi":"10.1021/acs.biochem.4c00385","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00385","url":null,"abstract":"Expression of camelid-derived, single-domain antibodies (V<sub>H</sub>Hs) within the cytoplasm of mammalian cells as “intrabodies” has opened up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein. The intrabody consists of two structurally defined V<sub>H</sub>Hs that target distinct epitopes on RT’s enzymatic subunit (RTA): V9E1 targets RTA’s P-stalk recruitment site, and V2A11 targets RTA’s active site. Resistance to RT conferred by the biparatopic V<sub>H</sub>H construct far exceeded that of either of the V<sub>H</sub>Hs alone and effectively inhibited all measurable RT-induced cytotoxicity <i>in vitro</i>. We propose that the targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.","PeriodicalId":501642,"journal":{"name":"Biochemistry","volume":"18 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142263155","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}
Pub Date : 2024-09-18DOI: 10.1021/acs.biochem.4c00401
Sayan Saha, Shankar Prasad Kanaujia
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.
{"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 (m<sup>5</sup>U) rRNA modifications frequently occur at U747 and U1939 (<i>Escherichia coli</i> numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of <i>S</i>-adenosyl-<span>l</span>-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 <i>Thermococcales</i> group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m<sup>5</sup>U (Arm<sup>5</sup>U) MTases. Among these, a tRNA-specific Arm<sup>5</sup>U MTase (<sub>Pab</sub>TrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm<sup>5</sup>U MTase from the hyperthermophilic archaeon <i>Pyrococcus horikoshii</i> (<i>Ph</i>RlmCD). 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 <i>Ph</i>RlmCD, 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 <i>Ph</i>RlmCD 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 Arm<sup>5</sup>U 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}
Pub Date : 2024-09-13DOI: 10.1021/acs.biochem.4c00150
Haoqian Liang, Youran Luo, Wilfred A. van der Donk
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.
对肽的 N 端和 C 端进行修饰可增强其稳定性，防止被外肽酶降解。许多多肽天然产物的生物合成途径都以酶修饰其末端为特征，这些酶可能是宝贵的生物催化剂库。兰特生物可可碱带有一个 N,N-二甲基化的 N-末端胺基。其生物合成基因簇编码推测的甲基转移酶 Cao4。在这项工作中，我们利用异源生产的肽作为模型底物，重组了该酶的活性，并根据标准化的anthipeptide命名法将其称为CaoSC。体外甲基化不同的anthipeptides揭示了CaoSC对底物的要求。该酶可接受不同长度和 C 端序列的肽，但在第二个位置需要脱氢丙氨酸或脱氢丁氨酸。CaoSC 介导的天然兰替霉素二甲基化使兰替霉素的抗菌活性略高于原生化合物。甲基化后活性和/或代谢稳定性的提高说明了 CaoSC 在治疗肽生物工程中的潜在应用前景。
{"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 <i>N</i>,<i>N</i>-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 CaoS<sub>C</sub> following standardized lanthipeptide nomenclature, using a heterologously produced peptide as the model substrate. In vitro methylation of diverse lanthipeptides revealed the substrate requirements of CaoS<sub>C</sub>. The enzyme accepts peptides of varying lengths and C-terminal sequences but requires dehydroalanine or dehydrobutyrine at the second position. CaoS<sub>C</sub>-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 CaoS<sub>C</sub> 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}
Pub Date : 2024-09-12DOI: 10.1021/acs.biochem.4c00379
Matthew Nava, Sebastian J. Rowe, Rebecca J. Taylor, Daniel Kahne, Daniel G. Nocera
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.
{"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 <b>l</b>ipo<b>p</b>olysaccharide <b>t</b>ransport proteins, <u>Lpt</u>B<sub>2</sub>FGCADE. 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<sup>–1</sup> 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}
Pub Date : 2024-09-12DOI: 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
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.
最近在烟酸芽孢杆菌（Bacillus niacini）中发现了一个负责降解烟酸（NA）的基因簇，目前正在对由此产生的酶的结构和功能进行评估，以确定途径中间体。该基因簇中的一个基因编码一种黄素单加氧酶（BnFMO），据推测该酶可催化羟化反应。对重组纯化的 BnFMO 进行的动力学分析表明，这种酶催化 2,6-DHNA 或 2,6-DHP 的羟化反应，后者是由 2,6-DHNA 的脱羧反应自发形成的。为了了解这种羟化反应的细节，我们采用了一种结合蛋白质 X 射线晶体学和低温电子显微镜（cryo-EM）的多模型方法确定了 BnFMO 的结构。在 2,6-DHP 存在的情况下，我们获得了配位的 BnFMO 冷冻电子显微镜结构，从而对潜在的催化残基进行了预测。结构数据表明，BnFMO 是三聚体，这在 A 类黄素单加氧酶中并不常见。在电子密度图和库仑势图中，都观察到了三聚体界面上的一个区域，该区域与脂质分子一致，并被模拟为脂质分子。高分辨率质谱分析表明，存在磷脂酰乙醇胺和磷脂酰甘油脂质的混合物。这些数据有助于深入了解烟酸芽孢杆菌有氧降解 NA 所特有的中心羟化反应的分子细节。
{"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 <i>Bacillus niacini</i> 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 <i>Bacillus niacini</i>.","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}
Pub Date : 2024-09-11DOI: 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
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.
α-l-(3′-2′)-三呋喃糖基核酸（TNA）可与自身配对，也可与 DNA 和 RNA 交叉配对，有望成为合成遗传学、诊断学和寡核苷酸疗法的工具。我们研究了人类反式离子合成（TLS）DNA聚合酶η（hPol η）在TNA修饰的模板链对面催化的体外引物插入和延伸反应。在 TNA-T（tT）对面，hPol η 主要插入 dAMP 和 dGMP，dTMP 和 dCMP 的效率较低，随后引物延伸为全长产物。hPol η 与模板 tT 和 O4-甲基 tT 的三元复合物在插入和延伸阶段的晶体结构表明，与 DNA 相比，TNA 的骨架更短，连接性也不同（分别为 3′ → 2′ 与 5′ → 3′），这导致了糖的取向、相邻磷酸间距和糖苷键方向的局部差异。引物末端胸腺嘧啶的 3′-OH 与进入的 dNTP 的 α-磷酸的平均距离为 3.4 Å，这与 hPol η 在 TNA 残基对面插入并延伸过去的情况一致。相反，hPol η-DNA-tTTP 三元复合物的晶体结构显示，引物末端的 3′-OH与 tTTP α-磷酸的距离太远，这与聚合酶无法结合 TNA 的情况一致。总之，我们的研究让人们更好地了解了 TLS DNA 聚合酶对模板中的非天然核苷酸和输入的三磷酸核苷的耐受性。
{"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-<span>l</span>-(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 <i>in vitro</i> 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 <i>O</i><sup>4</sup>-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 <i>O</i><sup>4</sup>-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and <i>O</i><sup>4</sup>-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}
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.
{"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}
Pub Date : 2024-09-10DOI: 10.1021/acs.biochem.4c00257
Kelsey Bohl, Sarah L. Wynia-Smith, Rachel A. Jones Lipinski, Brian C. Smith
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.
{"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<sup>+</sup> 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 <i>S</i>-nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO<sup>–</sup>) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO<sup>–</sup> (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<sup>–</sup> 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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：