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siRNA-Mimetic Ratiometric pH (sMiRpH) Probes for Improving Cell Delivery and mRNA Knockdown.
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-02-05 DOI: 10.1021/acschembio.4c00545
Madison R Herling, Lizeth Lopez Vazquez, Ivan J Dmochowski
{"title":"siRNA-Mimetic Ratiometric pH (sMiRpH) Probes for Improving Cell Delivery and mRNA Knockdown.","authors":"Madison R Herling, Lizeth Lopez Vazquez, Ivan J Dmochowski","doi":"10.1021/acschembio.4c00545","DOIUrl":"10.1021/acschembio.4c00545","url":null,"abstract":"<p><p>Second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2) were developed by hybridizing a 3'-FAM-labeled 2'-OMe RNA strand with a 3'-Cy5-labeled 25mer RNA strand. These duplexes demonstrated the silencing of cytoplasmic mRNA targets in HeLa cells as measured by RT-qPCR and supported by western blot analysis. Fluorescence intensity and lifetime measurements revealed that a single guanosine (G) positioned adjacent to FAM achieves substantial static quenching at pH 5, with additional collisional quenching rendering the dye almost nonemissive. A FAM-G π-π stacking interaction was evidenced by a red-shifted absorbance spectrum for FAM. Decreased quenching at near-neutral pH enhances the FAM dynamic range in the physiologic pH window and improves the differentiation in cells between endocytic entrapment and cytoplasmic release. Flow cytometric analysis of intracellular pH and uptake using sMiRpH-2 was corroborated by live cell confocal microscopy and found to be predictive of knockdown efficacy. A sMiRpH-2 probe successfully predicted the relative efficacy of two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of nonviral siRNA delivery vectors.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"309-320"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11854375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Scaffolding Activities of Pseudodeacetylase HDAC7.
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-02-05 DOI: 10.1021/acschembio.4c00753
Ishadi K M Kodikara, Mary Kay H Pflum
{"title":"Scaffolding Activities of Pseudodeacetylase HDAC7.","authors":"Ishadi K M Kodikara, Mary Kay H Pflum","doi":"10.1021/acschembio.4c00753","DOIUrl":"10.1021/acschembio.4c00753","url":null,"abstract":"<p><p>Histone deacetylase (HDAC) enzymes remove acetyl groups from acetyllysine-containing proteins, including nucleosomal histones to control gene expression. Beyond fundamental cell biology, HDAC activity is linked to various cancers, with many HDAC inhibitors developed as anticancer therapeutics. Among the 11 metal-dependent HDAC proteins, the four class IIa isoforms (HDAC4, 5, 7, and 9) are \"pseudodeacetylases\" without measurable enzymatic activity due to mutation of a catalytic tyrosine. Deacetylase-related activities of class IIa HDAC proteins are attributed to scaffolding functions, where recruitment of an active HDAC isoform leads to bound substrate deacetylation. Scaffolding of class IIa proteins beyond simple recruitment of an active HDAC is only starting to emerge. This review explores the various scaffolding roles of HDAC7, including recently reported acetylation-mediated reversible scaffolding, which is a form of acetyllysine-binding reader function. Studying the functional roles of HDAC7 will provide molecular insight into normal and pathological conditions, which could facilitate drug design.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"248-258"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254150","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
Workflow for E3 Ligase Ligand Validation for PROTAC Development.
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-02-11 DOI: 10.1021/acschembio.4c00812
Nebojša Miletić, Janik Weckesser, Thorsten Mosler, Rajeshwari Rathore, Marina E Hoffmann, Paul Gehrtz, Sarah Schlesiger, Ingo V Hartung, Nicola Berner, Stephanie Wilhelm, Juliane Müller, Bikash Adhikari, Václav Němec, Saran Aswathaman Sivashanmugam, Lewis Elson, Hanna Holzmann, Martin P Schwalm, Lasse Hoffmann, Kamal Rayees Abdul Azeez, Susanne Müller, Bernhard Kuster, Elmar Wolf, Ivan Đikić, Stefan Knapp
{"title":"Workflow for E3 Ligase Ligand Validation for PROTAC Development.","authors":"Nebojša Miletić, Janik Weckesser, Thorsten Mosler, Rajeshwari Rathore, Marina E Hoffmann, Paul Gehrtz, Sarah Schlesiger, Ingo V Hartung, Nicola Berner, Stephanie Wilhelm, Juliane Müller, Bikash Adhikari, Václav Němec, Saran Aswathaman Sivashanmugam, Lewis Elson, Hanna Holzmann, Martin P Schwalm, Lasse Hoffmann, Kamal Rayees Abdul Azeez, Susanne Müller, Bernhard Kuster, Elmar Wolf, Ivan Đikić, Stefan Knapp","doi":"10.1021/acschembio.4c00812","DOIUrl":"10.1021/acschembio.4c00812","url":null,"abstract":"<p><p>Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau ligase) E3 ligase ligands. However, the considerable size of the human E3 ligase family, newly developed E3 ligase ligands, and the favorable druggability of some E3 ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 ligase ligand efficiency for PROTAC development and the assessment of the corresponding \"degradable\" target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 ligase ligand negative controls, competition by kinase parent compounds, and neddylation and proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 ligase ligand systems for degrader development.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"507-521"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11851430/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143389493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Glycated α-Synuclein Renders Glial Cell Activation and Induces Degeneration of Dopaminergic Neurons: A Potential Implication for the Development of Parkinson’s Disease
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 DOI: 10.1021/acschembio.4c0077710.1021/acschembio.4c00777
Sayan Chatterjee, Arvind Verma, Harsh Thakkar, Ravi P. Shah* and Amit Khairnar*, 
{"title":"Glycated α-Synuclein Renders Glial Cell Activation and Induces Degeneration of Dopaminergic Neurons: A Potential Implication for the Development of Parkinson’s Disease","authors":"Sayan Chatterjee,&nbsp;Arvind Verma,&nbsp;Harsh Thakkar,&nbsp;Ravi P. Shah* and Amit Khairnar*,&nbsp;","doi":"10.1021/acschembio.4c0077710.1021/acschembio.4c00777","DOIUrl":"https://doi.org/10.1021/acschembio.4c00777https://doi.org/10.1021/acschembio.4c00777","url":null,"abstract":"<p >Accumulation of misfolded α-synuclein (α-Syn) leads to the formation of Lewy bodies and is a major hallmark of Parkinson’s disease (PD). The accumulation of α-Syn involves several post-translational modifications. Recently, though, glycation of α-Syn (advanced glycation end products) and activation of the receptor for advanced glycation end products (RAGE) have been linked to neuroinflammation, which leads to oxidative stress and accumulation of α-Syn. The present study aims to detect the effect of glycated α-Syn (gly-α-Syn)-induced synucleinopathy and loss of dopaminergic (DAergic) neurons in the development of PD. We isolated, purified, and prepared glycated recombinant human α-Syn using <span>d</span>-ribose. Gly-α-Syn was characterized by SDS-PAGE, intact mass analysis, and bottom-up peptide sequence through LC-HRMS/MS. The aggregation propensity of gly-α-Syn has been verified by morphological and shape analysis through Bio-AFM. The gly-α-Syn (2 μg/μL) was injected stereotaxically in the substantia nigra (SN) of ICR mice (3–4 months) and compared with the normal α-Syn, d ribose, and Tris-HCl/artificial CSF groups. 56 days postsurgery (DPS), an immunohistochemical examination was conducted to investigate gly-α-Syn-induced α-Syn accumulation, neuroinflammation, and neurodegeneration. The glycation of α-Syn led to the expression of transglutaminase 2 (TGM2), an enzyme that cross-linked with AGEs and may have caused the accumulation of α-Syn. Significant RAGE activation was also observed in gly-α-Syn, which might have induced glial cell activation, resulting in oxidative stress and, ultimately, apoptosis of dopaminergic neurons. It is important to note that TGM2, phosphorylated α-Syn, RAGE expression, and glial cell activation were only found in the gly-α-Syn group and not in the other groups. This suggests that gly-α-Syn plays a major role in synucleinopathy, neuroinflammation, and neurodegeneration. Overall, the present study demonstrated glycation of α-Syn as one of the important age-associated post-translational modifications that are involved in the degeneration of dopaminergic neurons, at least in a subset of the diabetic patients susceptible to developing PD.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":"20 3","pages":"632–645 632–645"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143667019","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
Novel Quinazoline Derivatives Inhibit Splicing of Fungal Group II Introns. 新型喹唑啉衍生物抑制真菌II族内含子剪接。
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-17 DOI: 10.1021/acschembio.4c00631
Olga Fedorova, Michelle Luo, G Erik Jagdmann, Michael C Van Zandt, Luke Sisto, Anna Marie Pyle
{"title":"Novel Quinazoline Derivatives Inhibit Splicing of Fungal Group II Introns.","authors":"Olga Fedorova, Michelle Luo, G Erik Jagdmann, Michael C Van Zandt, Luke Sisto, Anna Marie Pyle","doi":"10.1021/acschembio.4c00631","DOIUrl":"10.1021/acschembio.4c00631","url":null,"abstract":"<p><p>We report the discovery of small molecules that target the RNA tertiary structure of self-splicing group II introns and display potent antifungal activity against yeasts, including the major public health threat <i>Candida parapsilosis</i>. High-throughput screening efforts against a yeast group II intron resulted in an inhibitor class which was then synthetically optimized for enhanced inhibitory activity and antifungal efficacy. The most highly refined compounds in this series display strong, gene-specific antifungal activity against <i>C. parapsilosis</i>. This work demonstrates the utility of combining advanced RNA screening methodologies with medicinal chemistry pipelines to identify high-affinity ligands targeting RNA tertiary structures with important roles in human health and disease.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"378-385"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11851433/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142995968","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Discovery of DCAF16 Binders for Targeted Protein Degradation.
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-30 DOI: 10.1021/acschembio.4c00799
Miguel A Campos, Isabella A Riha, Chenlu Zhang, Chen Mozes, Karl A Scheidt, Xiaoyu Zhang
{"title":"Discovery of DCAF16 Binders for Targeted Protein Degradation.","authors":"Miguel A Campos, Isabella A Riha, Chenlu Zhang, Chen Mozes, Karl A Scheidt, Xiaoyu Zhang","doi":"10.1021/acschembio.4c00799","DOIUrl":"10.1021/acschembio.4c00799","url":null,"abstract":"<p><p>Conventional small-molecule drugs primarily operate by inhibiting protein function, but this approach is limited when proteins lack well-defined ligand-binding pockets. Targeted protein degradation (TPD) offers an alternative approach by harnessing cellular degradation pathways to eliminate specific proteins. Recent studies have expanded the potential of TPD by identifying additional E3 ligases, with DCAF16 emerging as a promising candidate for facilitating protein degradation through both proteolysis-targeting chimera (PROTAC) and molecular glue mechanisms. In this study, we revisited a previously reported compound and discovered that it covalently binds to DCAF16. We further optimized it into a FKBP12-targeting PROTAC, MC-25B. This PROTAC engages DCAF16 at cysteines C177-179, leading to the degradation of nuclear-localized FKBP12. We further demonstrated the versatility of this DCAF16 recruiter by degrading additional endogenous proteins. Compared to the first-generation DCAF16-based PROTAC, which was derived from a fragment electrophile, this DCAF16 recruiter-based PROTAC exhibits improved proteome-wide selectivity.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"479-488"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062111","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
YBX1 Modulates 8-Oxoguanine Recognition and Repair in DNA.
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-02-04 DOI: 10.1021/acschembio.4c00831
Xiaofang Zheng, Weiheng Kong, Xiaoxia Dai, Changjun You
{"title":"YBX1 Modulates 8-Oxoguanine Recognition and Repair in DNA.","authors":"Xiaofang Zheng, Weiheng Kong, Xiaoxia Dai, Changjun You","doi":"10.1021/acschembio.4c00831","DOIUrl":"10.1021/acschembio.4c00831","url":null,"abstract":"<p><p>8-Oxoguanine (8-oxoG) is not only a biomarker of oxidative DNA damage but also an epigenetic-like regulator in mammalian cells. The identification and characterization of 8-oxoG-binding proteins would be crucial for further understanding the biological consequences of 8-oxoG. Here, we identified human Y-box-binding protein 1 (YBX1) as a novel binding protein for 8-oxoG modification in DNA by using a quantitative proteomic approach. Moreover, we found that the deficiency of YBX1 can substantially decrease the cellular sensitivity to oxidative stress and facilitate the repair of 8-oxoG embedded in DNA. These findings provided new insight into the biological significance of the functional interplay between YBX1 and 8-oxoG modification in DNA.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"529-536"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187527","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
Multi-TACs: Targeting Solid Tumors with Multiple Immune Cell Co-engagers. multi - tac:用多种免疫细胞共接合物靶向实体肿瘤。
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-09 DOI: 10.1021/acschembio.4c00843
Yuxuan Zhang, Zijian Zhang, Feng Lin
{"title":"Multi-TACs: Targeting Solid Tumors with Multiple Immune Cell Co-engagers.","authors":"Yuxuan Zhang, Zijian Zhang, Feng Lin","doi":"10.1021/acschembio.4c00843","DOIUrl":"10.1021/acschembio.4c00843","url":null,"abstract":"<p><p>Multiple immune components in the complex and heterogeneous tumor-immune microenvironment (TIME) work cooperatively to promote or impede cancer immunotherapy. Synergistically co-managing multiple immune cells with single agents for advanced antitumor immunity remains desirable but challenging. This In Focus article introduces a triple orthogonal linker (T-Linker)-based multimodal targeting chimera (Multi-TAC) platform, enabling the single-agent-mediated tumor-targeted co-engagement of multiple immune cell types within TIME for potentiated immunotherapy.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"245-247"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142941419","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
Amide Internucleoside Linkages Suppress the MicroRNA-like Off-Target Activity of Short Interfering RNA. 酰胺核苷间键抑制短干扰RNA的microrna样脱靶活性。
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-15 DOI: 10.1021/acschembio.4c00824
Chandan Pal, Michael Richter, Jayamini Harasgama, Eriks Rozners
{"title":"Amide Internucleoside Linkages Suppress the MicroRNA-like Off-Target Activity of Short Interfering RNA.","authors":"Chandan Pal, Michael Richter, Jayamini Harasgama, Eriks Rozners","doi":"10.1021/acschembio.4c00824","DOIUrl":"10.1021/acschembio.4c00824","url":null,"abstract":"<p><p>RNA interference (RNAi) has rapidly matured as a novel therapeutic approach. In this field, chemical modifications have been critical to the clinical success of short interfering RNAs (siRNAs). Notwithstanding the significant advances, achieving robust durability and gene silencing in extrahepatic tissues, as well as reducing off-target effects of siRNA, are areas where chemical modifications can still improve siRNA performance. The present study developed the challenging synthesis of amide-linked guanosine dimers (G<sub>AM1</sub>G and G<sub>AM1</sub>A) and completed an \"amide walk\" one by one, systematically replacing every internucleoside phosphate with an amide linkage in a guide strand targeting the PIK3CB gene. Dual-luciferase and RT-qPCR assays in HeLa cells showed that, in a model system of unmodified siRNAs, the amide linkage at position 3 (between nucleosides 3 and 4) suppressed the cleavage of off-target YY1 and FADD mRNAs similarly to the industry gold standard modification glycol nucleic acid (GNA). These results suggest that amide linkages in the seed region have strong potential to improve the specificity of siRNAs by suppressing the microRNA-like off-target activity.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"522-528"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142981985","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
Identification of Key Amino Acids in the A Domains of Polymyxin Synthetase Responsible for 2,4-Diaminobutyric Acid Adenylation in Paenibacillus polymyxa NBRC3020 Strain. 多粘类芽孢杆菌NBRC3020多粘菌素合成酶A区2,4-二氨基丁酸腺苷化关键氨基酸的鉴定
IF 3.5 2区 生物学
ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-17 DOI: 10.1021/acschembio.4c00553
Mai Nemoto, Wataru Ando, Taichi Mano, Minjae Lee, Satoshi Yuzawa, Toshihisa Mizuno
{"title":"Identification of Key Amino Acids in the A Domains of Polymyxin Synthetase Responsible for 2,4-Diaminobutyric Acid Adenylation in <i>Paenibacillus polymyxa</i> NBRC3020 Strain.","authors":"Mai Nemoto, Wataru Ando, Taichi Mano, Minjae Lee, Satoshi Yuzawa, Toshihisa Mizuno","doi":"10.1021/acschembio.4c00553","DOIUrl":"10.1021/acschembio.4c00553","url":null,"abstract":"<p><p>Developing novel nonribosomal peptides (NRPs) requires a comprehensive understanding of the enzymes involved in their biosynthesis, particularly the substrate amino acid recognition mechanisms in the adenylation (A) domain. This study focused on the A domain responsible for adenylating l-2,4-diaminobutyric acid (l-Dab) within the synthetase of polymyxin, an NRP produced by <i>Paenibacillus polymyxa</i> NBRC3020. To date, investigations into recombinant proteins that selectively adenylate l-Dab─exploring substrate specificity and enzymatic activity parameters─have been limited to reports on A domains found in enzymes synthesizing l-Dab homopolymers (pldA from <i>S. celluloflavus</i> USE31 and pddA from <i>S. hindustanus</i> NBRC15115), which remain exceedingly rare. The polymyxin synthetase in NBRC3020 contains five A domains specific to l-Dab, distributed across five distinct modules (modules 1, 3, 4, 5, 8, and 9). In this study, we successfully obtained soluble A domain proteins from modules 1, 5, 8, and 9 by preparing module-specific recombinant proteins. These proteins were expressed in <i>E. coli</i> BAP-1, purified via Ni-affinity chromatography, and demonstrated high specificity for l-Dab. Through sequence homology analysis, three-dimensional structural modeling, docking simulations to estimate substrate-binding sites, and functional validation using alanine mutants, we identified Glu281 and Asp344 as critical residues for recognizing the side chain amino group of l-Dab, and Asp238 as essential for recognizing its main chain amino group in the A domain. Notably, these key residues were conserved not only across the A domains in modules 1, 5, 8, and 9 of <i>P. polymyxa</i> NBRC3020 but also in those of the <i>P. polymyxa</i> PKB1 strain, as confirmed by sequence homology analysis. Interestingly, in pldA and pddA, the key residues involved in recognizing the side-chain amino group of l-Dab, which are conserved among polymyxin synthetases of NBRC3020 and PKB1 strain, were not observed. This suggests a potentially different mechanism for l-Dab recognition.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"321-331"},"PeriodicalIF":3.5,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142995965","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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