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Bringing Histidine Phosphorylation into Light: Role of Chemical Tools.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-04 DOI: 10.1021/acschembio.5c00103
Solbee Choi, Shin Hyeon Lee, Jung-Min Kee

Histidine phosphorylation is a historically underexplored post-translational modification (PTM). Once deemed "elusive" due to its chemical lability, phosphohistidine (pHis) has recently come to light thanks to emerging chemical tools─including stable pHis analogs, pHis-specific antibodies, and tailored proteomics workflows─that enable its detection and functional analysis. Together, these innovations have led to a surge in the identification of pHis sites and raised awareness of their roles in both bacterial and mammalian systems. New assay systems have also facilitated the characterization of histidine kinases and phosphatases. This Review summarizes recent breakthroughs in pHis research tools, examines the limitations of current approaches, and outlines future tools needed to fully unravel the potential of histidine phosphorylation.

{"title":"Bringing Histidine Phosphorylation into Light: Role of Chemical Tools.","authors":"Solbee Choi, Shin Hyeon Lee, Jung-Min Kee","doi":"10.1021/acschembio.5c00103","DOIUrl":"https://doi.org/10.1021/acschembio.5c00103","url":null,"abstract":"<p><p>Histidine phosphorylation is a historically underexplored post-translational modification (PTM). Once deemed \"elusive\" due to its chemical lability, phosphohistidine (pHis) has recently come to light thanks to emerging chemical tools─including stable pHis analogs, pHis-specific antibodies, and tailored proteomics workflows─that enable its detection and functional analysis. Together, these innovations have led to a surge in the identification of pHis sites and raised awareness of their roles in both bacterial and mammalian systems. New assay systems have also facilitated the characterization of histidine kinases and phosphatases. This Review summarizes recent breakthroughs in pHis research tools, examines the limitations of current approaches, and outlines future tools needed to fully unravel the potential of histidine phosphorylation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Augmented Acyl-CoA Biosynthesis Promotes Resistance to TEAD Palmitoylation Site Inhibition.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-03 DOI: 10.1021/acschembio.5c00162
Kayla Nutsch, Marissa N Trujillo, Lirui Song, Michael A Erb, Jian Jeffery Chen, James J Galligan, Michael J Bollong

Activation of the YAP-TEAD transcriptional complex drives the growth of several cancer types and is a key resistance mechanism to targeted therapies. Accordingly, a host of pharmacological inhibitors to TEAD family paralogs have been developed, yet little is known as to the resistance mechanisms that might arise against this emerging therapeutic class. Here, we report that genetic augmentation of de novo coenzyme A biosynthesis desensitizes YAP-dependent cancer cells to treatment with TEAD inhibitors, an effect driven by increased levels of palmitoyl-CoA that outcompete drug for engagement of the lipid-binding pocket. This work uncovers a potential therapeutic resistance mechanism to TEAD palmitoylation site inhibition with implications for future combinatorial treatments in the clinic.

YAP-TEAD转录复合物的激活推动了几种癌症类型的生长,也是靶向疗法的一个关键耐药机制。因此,一系列针对 TEAD 家族旁系亲属的药理抑制剂已被开发出来,但人们对这一新兴治疗类别可能产生的抗药性机制却知之甚少。在这里,我们报告了通过基因增强辅酶A的生物合成可使依赖YAP的癌细胞对TEAD抑制剂脱敏,这种效应是由棕榈酰-CoA水平的增加所驱动的,棕榈酰-CoA可与药物竞争脂质结合口袋。这项研究揭示了 TEAD 棕榈酰化位点抑制的潜在治疗抗性机制,对未来临床中的组合疗法具有重要意义。
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引用次数: 0
Reconstitution and Characterization of Biosynthetic Machinery for Parageocin I, a Novel Thiazole-Rich Peptide from the Thermophilic Bacterium Parageobacillus caldoxylosilyticus.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-02 DOI: 10.1021/acschembio.4c00758
Ayane Yano, Hiroya Tomita, Kentaro Miyazaki, Kohsuke Honda

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are the representative microbial peptidyl secondary metabolites including the class of linear azol(in)e-containing peptides (LAPs). A substantial proportion of LAPs have been identified in mesophilic microorganisms, including actinomycetes. In this study, we report the biosynthetic reconstitution and characterization of parageocin I, a novel thiazole-rich LAP derived from the thermophilic bacterium Parageobacillus caldoxylosilyticus KH1-5 which exhibits optimal growth around 60 °C. The biosynthetic gene cluster (pgc) consists of four genes: pgcA, pgcB, pgcC, and pgcD, encoding the precursor peptide, dehydrogenase, YcaO family cyclodehydratase, and biosynthetic scaffold protein, respectively. The precursor peptide PgcA possesses 13 Cys and 2 Ser residues, with regularly repeated sequences interspaced between Cys residues. We first reconstituted the biosynthesis heterologously in Escherichia coli. Mass spectrometry analysis of the synthesized peptide, coupled with mutational analyses of the modified PgcA, revealed that the final product, designated as parageocin I, harbors 13 thiazole rings derived from the cyclization of Cys residues, while Ser residues remain intact. Furthermore, mutational studies of PgcA revealed three key principles governing heterocyclization by PgcC: (i) Cys is acceptable, but Ser and Thr are not; (ii) the presence of an acidic amino acid preceding Cys is not permissible; and (iii) a minimum of two amino acids must separate Cys residues. In addition, we successfully reconstituted the biosynthesis in vitro using the purified recombinant enzymes. This is the first report of LAP biosynthesis in thermophilic Bacillaceae, thereby expanding our understanding of not only LAPs but also secondary metabolism in thermophiles.

{"title":"Reconstitution and Characterization of Biosynthetic Machinery for Parageocin I, a Novel Thiazole-Rich Peptide from the Thermophilic Bacterium <i>Parageobacillus caldoxylosilyticus</i>.","authors":"Ayane Yano, Hiroya Tomita, Kentaro Miyazaki, Kohsuke Honda","doi":"10.1021/acschembio.4c00758","DOIUrl":"https://doi.org/10.1021/acschembio.4c00758","url":null,"abstract":"<p><p>Ribosomally synthesized and post-translationally modified peptides (RiPPs) are the representative microbial peptidyl secondary metabolites including the class of linear azol(in)e-containing peptides (LAPs). A substantial proportion of LAPs have been identified in mesophilic microorganisms, including actinomycetes. In this study, we report the biosynthetic reconstitution and characterization of parageocin I, a novel thiazole-rich LAP derived from the thermophilic bacterium <i>Parageobacillus caldoxylosilyticus</i> KH1-5 which exhibits optimal growth around 60 °C. The biosynthetic gene cluster (<i>pgc</i>) consists of four genes: <i>pgcA</i>, <i>pgcB</i>, <i>pgcC</i>, and <i>pgcD</i>, encoding the precursor peptide, dehydrogenase, YcaO family cyclodehydratase, and biosynthetic scaffold protein, respectively. The precursor peptide PgcA possesses 13 Cys and 2 Ser residues, with regularly repeated sequences interspaced between Cys residues. We first reconstituted the biosynthesis heterologously in <i>Escherichia coli</i>. Mass spectrometry analysis of the synthesized peptide, coupled with mutational analyses of the modified PgcA, revealed that the final product, designated as parageocin I, harbors 13 thiazole rings derived from the cyclization of Cys residues, while Ser residues remain intact. Furthermore, mutational studies of PgcA revealed three key principles governing heterocyclization by PgcC: (i) Cys is acceptable, but Ser and Thr are not; (ii) the presence of an acidic amino acid preceding Cys is not permissible; and (iii) a minimum of two amino acids must separate Cys residues. In addition, we successfully reconstituted the biosynthesis in vitro using the purified recombinant enzymes. This is the first report of LAP biosynthesis in thermophilic Bacillaceae, thereby expanding our understanding of not only LAPs but also secondary metabolism in thermophiles.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Noninvasive Bioluminescence Imaging of Serum Albumins in Living Mice.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-02 DOI: 10.1021/acschembio.4c00740
Sung-Bae Kim, Genta Kamiya, Tadaomi Furuta, Nobuo Kitada, Suresh Thangudu, Arutselvan Natarajan, Shojiro A Maki, Ramasamy Paulmurugan

Bioluminescence (BL) is an emerging optical readout that has been extensively used in various bioassays and molecular imaging systems. In this study, we present the bioanalytical application of marine luciferins as an excellent optical indicator for noninvasive imaging of serum albumins. We synthesized 30 kinds of regioisomeric coelenterazine (CTZ) analogs and investigated their specificities for major serum proteins from various species. The results found that some of the CTZ analogs exhibited surprisingly specific optical signals upon binding with the serum albumins. These CTZ analogs showed diverse emission spectra ranging from 495 to 558 nm according to the albumin species used acting as pseudoluciferases. The selective albumin indicators, TS1 and TS2, exhibited long and linear dose-response curves and were sensitive enough to determine clinically normal and abnormal (microalbuminuria) ranges of albumins in saliva and urine. The sensitivity of this assay is superior to that of the conventional Bromocresol purple (BCP) method. We further demonstrated the advantages of the albumin indicators through noninvasive imaging of liver-albumin in vivo in living mice. The in vivo and ex vivo imaging results confirmed that the CTZ analog TS2 can sensitively image the liver-albumin in vivo with high signal-to-background ratio. This study paves a new way to make use of CTZ analogs for noninvasive albumin imaging and conceptualizes the pseudoluciferase-based imaging. The distinct in vivo imaging of serum albumins can potentially aid clinicians in providing insight into patients' liver function and other vital factors needed for whole-body homeostasis.

{"title":"Noninvasive Bioluminescence Imaging of Serum Albumins in Living Mice.","authors":"Sung-Bae Kim, Genta Kamiya, Tadaomi Furuta, Nobuo Kitada, Suresh Thangudu, Arutselvan Natarajan, Shojiro A Maki, Ramasamy Paulmurugan","doi":"10.1021/acschembio.4c00740","DOIUrl":"https://doi.org/10.1021/acschembio.4c00740","url":null,"abstract":"<p><p>Bioluminescence (BL) is an emerging optical readout that has been extensively used in various bioassays and molecular imaging systems. In this study, we present the bioanalytical application of marine luciferins as an excellent optical indicator for noninvasive imaging of serum albumins. We synthesized 30 kinds of regioisomeric coelenterazine (CTZ) analogs and investigated their specificities for major serum proteins from various species. The results found that some of the CTZ analogs exhibited surprisingly specific optical signals upon binding with the serum albumins. These CTZ analogs showed diverse emission spectra ranging from 495 to 558 nm according to the albumin species used acting as pseudoluciferases. The selective albumin indicators, <b>TS1</b> and <b>TS2</b>, exhibited long and linear dose-response curves and were sensitive enough to determine clinically normal and abnormal (microalbuminuria) ranges of albumins in saliva and urine. The sensitivity of this assay is superior to that of the conventional Bromocresol purple (BCP) method. We further demonstrated the advantages of the albumin indicators through noninvasive imaging of liver-albumin <i>in vivo</i> in living mice. The <i>in vivo</i> and <i>ex vivo</i> imaging results confirmed that the CTZ analog <b>TS2</b> can sensitively image the liver-albumin <i>in vivo</i> with high signal-to-background ratio. This study paves a new way to make use of CTZ analogs for noninvasive albumin imaging and conceptualizes the pseudoluciferase-based imaging. The distinct <i>in vivo</i> imaging of serum albumins can potentially aid clinicians in providing insight into patients' liver function and other vital factors needed for whole-body homeostasis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Engineering the Specificity of Acetyl-CoA Synthetase for Diverse Acyl-CoA Thioester Generation.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-02 DOI: 10.1021/acschembio.5c00014
Jared R Cossin, Thaddeus Q Paulsel, Kim Castelli, Breck Wcisel, Alexandra A Malico, Gavin J Williams

CoA thioesters are valuable intermediates in numerous biosynthetic routes and metabolic processes. However, diversifying these compounds and their corresponding downstream products hinges on broadening the promiscuity of CoA ligases that produce them or using additional enzymes to functionalize them. Here, the inherent promiscuity of an acyl-CoA ligase from Pseudomonas chlororaphis was probed with carboxylic acids of varying sizes and functionality. The enzyme was engineered to improve its activity with a diverse panel of acyl-CoA thioesters, including halogenated and oxidized acids, that can be used in downstream biosynthetic production strategies. To demonstrate the utility of the engineered enzyme, a subset of the substrates was leveraged for the complete in situ biosynthesis of a small panel of pyrones via a portion of the archetypal polyketide synthase (PKS), 6-deoxyerythronolide B synthase (DEBS). This approach supports probing the promiscuity of polyketide biosynthesis and the diversification of natural product scaffolds.

{"title":"Engineering the Specificity of Acetyl-CoA Synthetase for Diverse Acyl-CoA Thioester Generation.","authors":"Jared R Cossin, Thaddeus Q Paulsel, Kim Castelli, Breck Wcisel, Alexandra A Malico, Gavin J Williams","doi":"10.1021/acschembio.5c00014","DOIUrl":"https://doi.org/10.1021/acschembio.5c00014","url":null,"abstract":"<p><p>CoA thioesters are valuable intermediates in numerous biosynthetic routes and metabolic processes. However, diversifying these compounds and their corresponding downstream products hinges on broadening the promiscuity of CoA ligases that produce them or using additional enzymes to functionalize them. Here, the inherent promiscuity of an acyl-CoA ligase from <i>Pseudomonas chlororaphis</i> was probed with carboxylic acids of varying sizes and functionality. The enzyme was engineered to improve its activity with a diverse panel of acyl-CoA thioesters, including halogenated and oxidized acids, that can be used in downstream biosynthetic production strategies. To demonstrate the utility of the engineered enzyme, a subset of the substrates was leveraged for the complete <i>in situ</i> biosynthesis of a small panel of pyrones via a portion of the archetypal polyketide synthase (PKS), 6-deoxyerythronolide B synthase (DEBS). This approach supports probing the promiscuity of polyketide biosynthesis and the diversification of natural product scaffolds.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Epitope Mapping of Antibody Binding Sites on Human Arginase-1.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-04-01 DOI: 10.1021/acschembio.4c00692
Jaime Fernández de Santaella, Nikolaj G Koch, Lorenz Widmer, Michael A Nash

Epitope mapping is crucial for understanding immunological responses to protein therapeutics. Here, we combined genetic code expansion and bacterial surface display to incorporate S-allylcysteine (SAC) into human arginase-1 (hArg1) via Methanococcoides burtonii pyrrolysyl-tRNA synthetase. Using an amber codon deep mutational scanning and sequencing workflow, we mapped SAC incorporation efficiency across the hArg1 sequence, providing insights into structural and sequence dependencies of noncanonical amino acid incorporation. We used mutually bioorthogonal allyl/tetrazine and azide/DBCO chemistries to achieve site-specific PEGylation and fluorescent labeling of hArg1, revealing insights into SAC side chain reactivity and solvent accessibility of residues in hArg1. This system was further applied to determine the binding epitope of a monoclonal antibody on the surface of hArg1, providing high-resolution data on the impact of PEGylation residue position on antibody binding. Our method produces high dimensional data of noncanonical amino acid incorporation efficiency, site-specific functionalization enabled by mutually bioorthogonal chemistries, and epitope mapping of therapeutic proteins.

{"title":"Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Epitope Mapping of Antibody Binding Sites on Human Arginase-1.","authors":"Jaime Fernández de Santaella, Nikolaj G Koch, Lorenz Widmer, Michael A Nash","doi":"10.1021/acschembio.4c00692","DOIUrl":"https://doi.org/10.1021/acschembio.4c00692","url":null,"abstract":"<p><p>Epitope mapping is crucial for understanding immunological responses to protein therapeutics. Here, we combined genetic code expansion and bacterial surface display to incorporate S-allylcysteine (SAC) into human arginase-1 (hArg1) via <i>Methanococcoides burtonii</i> pyrrolysyl-tRNA synthetase. Using an amber codon deep mutational scanning and sequencing workflow, we mapped SAC incorporation efficiency across the hArg1 sequence, providing insights into structural and sequence dependencies of noncanonical amino acid incorporation. We used mutually bioorthogonal allyl/tetrazine and azide/DBCO chemistries to achieve site-specific PEGylation and fluorescent labeling of hArg1, revealing insights into SAC side chain reactivity and solvent accessibility of residues in hArg1. This system was further applied to determine the binding epitope of a monoclonal antibody on the surface of hArg1, providing high-resolution data on the impact of PEGylation residue position on antibody binding. Our method produces high dimensional data of noncanonical amino acid incorporation efficiency, site-specific functionalization enabled by mutually bioorthogonal chemistries, and epitope mapping of therapeutic proteins.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","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 Biochemical Insights into Lignin-Oxidizing Activity of Bacterial Peroxidases against Soluble Substrates and Kraft Lignin.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.4c00788
Zahra Choolaei, Anna N Khusnutdinova, Tatiana Skarina, Peter Stogios, Patrick Diep, Sofia Lemak, Elizabeth A Edwards, Alexei Savchenko, Alexander F Yakunin

Great interest has recently been drawn to the production of value-added products from lignin; however, its recalcitrance and high chemical complexity have made this challenging. Dye-decolorizing peroxidases and catalase-peroxidases are among the enzymes that are recognized to play important roles in environmental lignin oxidation. However, bacterial lignin-oxidizing enzymes remain less characterized compared to related proteins from fungi. In this study, screening of 18 purified bacterial peroxidases against the general chromogenic substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) revealed the presence of peroxidase activity in all proteins. Agarose plate-based screens with kraft lignin identified detectable and high lignin oxidation activity in 15 purified proteins. Crystal structures were determined for the DyP-type peroxidases FC2591 from Frankia casuarinae, PF3257 from Pseudomonas fluorescens, and PR9465 from Pseudomonas rhizosphaerae. The structures revealed the presence of hemes with bound oxygens coordinated by conserved His, Arg, and Asp residues as well as three molecular tunnels connecting the heme with the protein surface. Structure-based site-directed mutagenesis of FC2591 identified at least five active site residues as essential for oxidase activity against both ABTS and lignin, whereas the S370A mutant protein showed a three- to 4-fold activity increase with both substrates. HPLC analysis of reaction products of the wild-type FC2591 and S370A mutant proteins with the model lignin dimer guaiacylglycerol-β-guaiacyl ether and kraft lignin revealed the formation of products consistent with the radical coupling of the reaction intermediates. Thus, this study identified novel bacterial heme peroxidases with lignin oxidation activity and provided further insights into our understanding of these enzymes.

{"title":"Structural and Biochemical Insights into Lignin-Oxidizing Activity of Bacterial Peroxidases against Soluble Substrates and Kraft Lignin.","authors":"Zahra Choolaei, Anna N Khusnutdinova, Tatiana Skarina, Peter Stogios, Patrick Diep, Sofia Lemak, Elizabeth A Edwards, Alexei Savchenko, Alexander F Yakunin","doi":"10.1021/acschembio.4c00788","DOIUrl":"https://doi.org/10.1021/acschembio.4c00788","url":null,"abstract":"<p><p>Great interest has recently been drawn to the production of value-added products from lignin; however, its recalcitrance and high chemical complexity have made this challenging. Dye-decolorizing peroxidases and catalase-peroxidases are among the enzymes that are recognized to play important roles in environmental lignin oxidation. However, bacterial lignin-oxidizing enzymes remain less characterized compared to related proteins from fungi. In this study, screening of 18 purified bacterial peroxidases against the general chromogenic substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) revealed the presence of peroxidase activity in all proteins. Agarose plate-based screens with kraft lignin identified detectable and high lignin oxidation activity in 15 purified proteins. Crystal structures were determined for the DyP-type peroxidases FC2591 from <i>Frankia casuarinae</i>, PF3257 from <i>Pseudomonas fluorescens</i>, and PR9465 from <i>Pseudomonas rhizosphaerae</i>. The structures revealed the presence of hemes with bound oxygens coordinated by conserved His, Arg, and Asp residues as well as three molecular tunnels connecting the heme with the protein surface. Structure-based site-directed mutagenesis of FC2591 identified at least five active site residues as essential for oxidase activity against both ABTS and lignin, whereas the S370A mutant protein showed a three- to 4-fold activity increase with both substrates. HPLC analysis of reaction products of the wild-type FC2591 and S370A mutant proteins with the model lignin dimer guaiacylglycerol-β-guaiacyl ether and kraft lignin revealed the formation of products consistent with the radical coupling of the reaction intermediates. Thus, this study identified novel bacterial heme peroxidases with lignin oxidation activity and provided further insights into our understanding of these enzymes.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
MYC-Targeting PROTACs Lead to Bimodal Degradation and N-Terminal Truncation.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.4c00864
Shelton R Boyd, Srinivas Chamakuri, Alexander J Trostle, Hu Chen, Zhandong Liu, Antrix Jian, Jian Wang, Anna Malovannaya, Damian W Young

MYC is a master regulatory transcription factor whose sustained dysregulation promotes the initiation and maintenance of numerous cancers. While MYC is a regarded as a potenial therapeutic target in cancer, its intrinsically disordered structure has proven to be a formidable barrier toward the development of highly effective small molecule inhibitors. We rationalized that proteolysis targeting chimeras (PROTACs), which might accomplish the targeted degradation of MYC, would achieve more potent cell killing in MYC-driven cancer cells than reversible inhibitors. PROTACs are bifunctional small molecules designed to produce a ternary complex between a target protein and an E3 ligase leading the target's ubiquitination and degradation by the 26S proteasome. We generated PROTAC MTP3 based on modifications of the previously reported MYC-targeting compound KJ-Pyr-9. We found that MTP3 depletes endogenous full-length MYC proteins and uniquely induces increasing levels of a functional, N-terminally truncated MYC species, tMYC. Furthermore, MTP3 perturbs cellular MYC levels in favor of a tMYC-dominated state whose gene regulatory landscape is not significantly altered compared to that of wild type MYC. Moreover, although it lacks ∼10 kDa of MYC's N-terminal transactivation domain, tMYC is sufficient to maintain an oncogenic proliferative state. Our results highlight the complexities of proximity-inducing compounds against highly regulated and conformationally dynamic protein targets such as MYC and indicate that PROTACs can induce alternative outcomes beyond target protein degradation.

{"title":"MYC-Targeting PROTACs Lead to Bimodal Degradation and N-Terminal Truncation.","authors":"Shelton R Boyd, Srinivas Chamakuri, Alexander J Trostle, Hu Chen, Zhandong Liu, Antrix Jian, Jian Wang, Anna Malovannaya, Damian W Young","doi":"10.1021/acschembio.4c00864","DOIUrl":"https://doi.org/10.1021/acschembio.4c00864","url":null,"abstract":"<p><p>MYC is a master regulatory transcription factor whose sustained dysregulation promotes the initiation and maintenance of numerous cancers. While MYC is a regarded as a potenial therapeutic target in cancer, its intrinsically disordered structure has proven to be a formidable barrier toward the development of highly effective small molecule inhibitors. We rationalized that proteolysis targeting chimeras (PROTACs), which might accomplish the targeted degradation of MYC, would achieve more potent cell killing in MYC-driven cancer cells than reversible inhibitors. PROTACs are bifunctional small molecules designed to produce a ternary complex between a target protein and an E3 ligase leading the target's ubiquitination and degradation by the 26S proteasome. We generated PROTAC MTP3 based on modifications of the previously reported MYC-targeting compound KJ-Pyr-9. We found that MTP3 depletes endogenous full-length MYC proteins and uniquely induces increasing levels of a functional, N-terminally truncated MYC species, tMYC. Furthermore, MTP3 perturbs cellular MYC levels in favor of a tMYC-dominated state whose gene regulatory landscape is not significantly altered compared to that of wild type MYC. Moreover, although it lacks ∼10 kDa of MYC's N-terminal transactivation domain, tMYC is sufficient to maintain an oncogenic proliferative state. Our results highlight the complexities of proximity-inducing compounds against highly regulated and conformationally dynamic protein targets such as MYC and indicate that PROTACs can induce alternative outcomes beyond target protein degradation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143726998","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Chitinivorax: The New Kid on the Block of Bacterial 2-Alkyl-4(1H)-quinolone Producers.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.5c00046
Viktoriia Savchenko, Xiaoqian Annie Yu, Martin F Polz, Thomas Böttcher

2-Alkyl-4(1H)-quinolones play a key role in bacterial communication, regulating biofilm formation, and virulence. Their antimicrobial properties also support bacterial survival and interspecies competition in microbial communities. In addition to the human pathogen Pseudomonas aeruginosa various species of Burkholderia and Pseudoalteromonas are known to produce 2-alkyl-4(1H)-quinolones. However, the evolutionary relationships of their biosynthetic gene clusters remain largely unexplored. To address this, we investigated the phylogeny of 2-alkyl-4(1H)-quinolone biosynthetic gene clusters, leading to the discovery of Chitinivorax as a fourth genus capable of producing 2-alkyl-4(1H)-quinolones, expanding our knowledge of the diversity of bacteria involved in quinolone-biosynthesis.

{"title":"Chitinivorax: The New Kid on the Block of Bacterial 2-Alkyl-4(1<i>H</i>)-quinolone Producers.","authors":"Viktoriia Savchenko, Xiaoqian Annie Yu, Martin F Polz, Thomas Böttcher","doi":"10.1021/acschembio.5c00046","DOIUrl":"https://doi.org/10.1021/acschembio.5c00046","url":null,"abstract":"<p><p>2-Alkyl-4(1<i>H</i>)-quinolones play a key role in bacterial communication, regulating biofilm formation, and virulence. Their antimicrobial properties also support bacterial survival and interspecies competition in microbial communities. In addition to the human pathogen <i>Pseudomonas aeruginosa</i> various species of <i>Burkholderia</i> and <i>Pseudoalteromonas</i> are known to produce 2-alkyl-4(1<i>H</i>)-quinolones. However, the evolutionary relationships of their biosynthetic gene clusters remain largely unexplored. To address this, we investigated the phylogeny of 2-alkyl-4(1<i>H</i>)-quinolone biosynthetic gene clusters, leading to the discovery of <i>Chitinivorax</i> as a fourth genus capable of producing 2-alkyl-4(1<i>H</i>)-quinolones, expanding our knowledge of the diversity of bacteria involved in quinolone-biosynthesis.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Formation of the i-motif Structures by Human Telomeric c-Rich Sequences d(CCCTAA)n and Its Recognition by Bisbenzylisoquinoline Alkaloids.
IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-03-27 DOI: 10.1021/acschembio.4c00844
Junliu Huang, Zexuan Lin, Jishun Yang, Huining Tang, Yang Yang, Yi Tang, Feixian Luo, Wenshu Wang, Xiaojie Cui

The human telomeric repeat CCCTAA has been reported to form a higher-order structure called an intercalated motif (i-motif) that plays important roles in telomere function and telomerase activity regulation, and small molecule ligands targeting human telomeric i-motif (hTelo-iM) is a promising therapeutic strategy for cancer treatment, yet the i-motif folding pattern of long CCCTAA repeats and the hTelo-iM ligand screening have not been studied extensively. In this study, we systematically investigated the i-motif structures formed by four and eight telomeric C-rich repeats d(CCCTAA)4 (hTeloC-24mer) and d(CCCTAA)8 (hTeloC-48mer) under varied conditions and found that the long hTeloC-48mer probably forms unstacked tandem i-motif consisting of two hTeloC-24mer i-motif monomers under near physiological conditions. Moreover, natural bisbenzylisoquinoline (BBI) alkaloids, isofangchinoline, fangchinoline, cepharanthine, and tetrandrine, were screened from 33 natural small molecules to effectively disrupt and destabilize the hTelo-iM structures mainly through major groove hydrogen bonding and van der Waals interactions. Further, telomerase repeated amplification protocol (TRAP) assay suggested that the selected BBI alkaloids can inhibit the telomere extension by telomerase. These findings provide a theoretical basis for further telomere structure research as well as a novel class of natural small molecule compounds regulating the hTelo-iM structure and telomerase activity, which may contribute to the anticancer drug design and strategy development taking the hTelo-iM as a target.

{"title":"Formation of the i-motif Structures by Human Telomeric c-Rich Sequences d(CCCTAA)n and Its Recognition by Bisbenzylisoquinoline Alkaloids.","authors":"Junliu Huang, Zexuan Lin, Jishun Yang, Huining Tang, Yang Yang, Yi Tang, Feixian Luo, Wenshu Wang, Xiaojie Cui","doi":"10.1021/acschembio.4c00844","DOIUrl":"https://doi.org/10.1021/acschembio.4c00844","url":null,"abstract":"<p><p>The human telomeric repeat CCCTAA has been reported to form a higher-order structure called an intercalated motif (i-motif) that plays important roles in telomere function and telomerase activity regulation, and small molecule ligands targeting human telomeric i-motif (hTelo-iM) is a promising therapeutic strategy for cancer treatment, yet the i-motif folding pattern of long CCCTAA repeats and the hTelo-iM ligand screening have not been studied extensively. In this study, we systematically investigated the i-motif structures formed by four and eight telomeric C-rich repeats d(CCCTAA)<sub>4</sub> (hTeloC-24mer) and d(CCCTAA)<sub>8</sub> (hTeloC-48mer) under varied conditions and found that the long hTeloC-48mer probably forms unstacked tandem i-motif consisting of two hTeloC-24mer i-motif monomers under near physiological conditions. Moreover, natural bisbenzylisoquinoline (BBI) alkaloids, isofangchinoline, fangchinoline, cepharanthine, and tetrandrine, were screened from 33 natural small molecules to effectively disrupt and destabilize the hTelo-iM structures mainly through major groove hydrogen bonding and van der Waals interactions. Further, telomerase repeated amplification protocol (TRAP) assay suggested that the selected BBI alkaloids can inhibit the telomere extension by telomerase. These findings provide a theoretical basis for further telomere structure research as well as a novel class of natural small molecule compounds regulating the hTelo-iM structure and telomerase activity, which may contribute to the anticancer drug design and strategy development taking the hTelo-iM as a target.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717569","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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