ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-02-21DOI: 10.1021/acschembio.4c00576
Marta Majewska, Maja Hanić, Rabea Bartölke, Jessica Schmidt, Justyna Bożek, Luca Gerhards, Henrik Mouritsen, Karl-Wilhelm Koch, Ilia A Solov'yov, Izabella Brand
{"title":"European Robin Cryptochrome-4a Associates with Lipid Bilayers in an Ordered Manner, Fulfilling a Molecular-Level Condition for Magnetoreception.","authors":"Marta Majewska, Maja Hanić, Rabea Bartölke, Jessica Schmidt, Justyna Bożek, Luca Gerhards, Henrik Mouritsen, Karl-Wilhelm Koch, Ilia A Solov'yov, Izabella Brand","doi":"10.1021/acschembio.4c00576","DOIUrl":"10.1021/acschembio.4c00576","url":null,"abstract":"<p><p>Since the middle of the 20th century, long-distance avian migration has been known to rely partly on geomagnetic field. However, the underlying sensory mechanism is still not fully understood. Cryptochrome-4a (ErCry4a), found in European robin (<i>Erithacus rubecula</i>), a night-migratory songbird, has been suggested to be a magnetic sensory molecule. It is sensitive to external magnetic fields via the so-called radical-pair mechanism. ErCry4a is primarily located in the outer segments of the double-cone photoreceptor cells in the eye, which contain stacked and highly ordered membranes that could facilitate the anisotropic attachment of ErCry4a needed for magnetic compass sensing. Here, we investigate possible interactions of ErCry4a with a model membrane that mimics the lipid composition of outer segments of vertebrate photoreceptor cells using experimental and computational approaches. Experimental results show that the attachment of ErCry4a to the membrane could be controlled by the physical state of lipid molecules (average area per lipid) in the outer leaflet of the lipid bilayer. Furthermore, polarization modulation infrared reflection absorption spectroscopy allowed us to determine the conformation, motional freedom, and average orientation of the α-helices in ErCry4a in a membrane-associated state. Atomistic molecular dynamics studies supported the experimental results. <i>A</i> ∼ 1000 kcal mol<sup>-1</sup> decrease in the interaction energy as a result of ErCry4a membrane binding was determined compared to cases where no protein binding to the membrane occurred. At the molecular level, the binding seems to involve negatively charged carboxylate groups of the phosphoserine lipids and the C-terminal residues of ErCry4a. Our study reveals a potential direct interaction of ErCry4a with the lipid membrane and discusses how this binding could be an essential step for ErCry4a to propagate a magnetic signal further and thus fulfill a role as a magnetoreceptor.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"592-606"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934094/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143466606","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-03-04DOI: 10.1021/acschembio.4c00835
Jinmin Liu, Yimon Aye
{"title":"Tools to Dissect Lipid Droplet Regulation, Players, and Mechanisms.","authors":"Jinmin Liu, Yimon Aye","doi":"10.1021/acschembio.4c00835","DOIUrl":"10.1021/acschembio.4c00835","url":null,"abstract":"<p><p>Spurred by the authors' own recent discovery of reactive metabolite-regulated nexuses involving lipid droplets (LDs), this perspective discusses the latest knowledge and multifaceted approaches toward deconstructing the function of these dynamic organelles, LD-associated localized signaling networks, and protein players. Despite accumulating knowledge surrounding protein families and pathways of conserved importance for LD homeostasis surveillance and maintenance across taxa, much remains to be understood at the molecular level. In particular, metabolic stress-triggered contextual changes in LD-proteins' localized functions, crosstalk with other organelles, and feedback signaling loops and how these are specifically rewired in disease states remain to be illuminated with spatiotemporal precision. We hope this perspective promotes an increased interest in these essential organelles and innovations of new tools and strategies to better understand context-specific LD regulation critical for organismal health.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"539-552"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539473","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-02-15DOI: 10.1021/acschembio.4c00849
Eva Schaller, Julian Hofmann, Pamela Maher, Christian Stigloher, Michael Decker
{"title":"Visualizing Intracellular Localization of Natural-Product-Based Chemical Probes Using Click-Correlative Light and Electron Microscopy.","authors":"Eva Schaller, Julian Hofmann, Pamela Maher, Christian Stigloher, Michael Decker","doi":"10.1021/acschembio.4c00849","DOIUrl":"10.1021/acschembio.4c00849","url":null,"abstract":"<p><p>Flavonoids such as sterubin and fisetin─and derivatives thereof─show strong neuroprotective effects <i>in vitro</i> as well as <i>in vivo</i>, combined with negligible toxicity and can therefore be considered novel treatment options for neurodegenerative diseases such as Alzheimer's disease. However, their subcellular locations responsible for neuroprotection and exact modes of action still remain unclear. Here, we present chemical probes based on both flavonoids sterubin and fisetin that were utilized in fluorescence microscopy and click-correlative light and electron microscopy to detect and visualize the localization of specific intracellular targets. We successfully adapted the workflow of correlative light and electron microscopy to a click-chemistry-based approach in a murine hippocampal cell line (HT22) on ultrathin resin sections making visualization of a small molecule for the first time possible in this setup. Utilizing this newly adapted technique, we could demonstrate that sterubin and fisetin show specific enrichment in the endoplasmic reticulum.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"721-730"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143424617","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-02-12DOI: 10.1021/acschembio.4c00582
Makwana Milee, Shanavas P Greeshma, Waghela Deeksha, Eerappa Rajakumara
{"title":"Regulatory and Catalytic Domains of Poly(ADP-ribose) Polymerases Cross-Complement for DNA-Break-Dependent Allosteric Stimulation of Catalytic Activity.","authors":"Makwana Milee, Shanavas P Greeshma, Waghela Deeksha, Eerappa Rajakumara","doi":"10.1021/acschembio.4c00582","DOIUrl":"10.1021/acschembio.4c00582","url":null,"abstract":"<p><p>Allosteric regulation is achieved by regulatory domains that sense stimuli and induce conformational changes in the functional domain that performs the catalytic activity of the enzyme. Poly-ADP-ribose polymerases (PARPs) are modular enzymes present across all domains of life including Archaea, Bacteria, and Eukarya. A typical domain architecture of PARPs consists of a conserved C-terminal catalytic domain (CAT) associated with multiple distinct N-terminal sensory and/or regulatory domains which together serve as regulatory region (REG). In this study, we investigated whether REG of different orthologs and paralogs of PARPs from mammals (hPARP1 and hPARP2), plants (atPARP2), and bacteria (haPARP) can assemble with CAT of each other to generate functional chimeric assemblies. We have employed qualitative and quantitative enzyme activity assays along with binding studies to examine these in vitro chimeric assemblies. The <i>cis</i>-complemented REG and CAT of hPARP2 exhibited micromolar binding affinity, suggesting that these domains can interact independent of allosteric ligands. Also, our results show that REG and CAT of PARP proteins can assemble in a functionally active conformation in the presence of DNA implying that REG and CAT are not required to be present on a single polypeptide for catalytic activity stimulation. Interestingly, only CAT of atPARP2 displayed functional complementation with REG of the other studied PARPs. Conversely, REG of hPARP1 and atPARP2 failed to <i>cross</i>-complement CAT of other PARPs while REG of hPARP2 showed robust <i>cross</i>-complementation. Our novel studies on chimeric PARP assemblies can be developed as a powerful synthetic biology tool to interrogate and control their activities in living cells. In addition, by co-engineering non-complementing REG and CAT domains of different PARPs, new functional chimeric PARPs can be developed for selective allosteric ligand-dependent regulation of PARP systems. Furthermore, our study can facilitate the understanding of the coevolution of REG and CAT domains in PARP enzymes.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"607-619"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143397478","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-03-10DOI: 10.1021/acschembio.4c00878
Cole Emanuelson, Yuta Naro, Olivia Shade, Melinda Liu, Sagar D Khare, Alexander Deiters
{"title":"Rational Design of Stapled Covalent Peptide Modifiers of Oncoprotein E6 from Human Papillomavirus.","authors":"Cole Emanuelson, Yuta Naro, Olivia Shade, Melinda Liu, Sagar D Khare, Alexander Deiters","doi":"10.1021/acschembio.4c00878","DOIUrl":"10.1021/acschembio.4c00878","url":null,"abstract":"<p><p>Human Papillomavirus (HPV) is linked to multiple cancers, most significantly cervical cancer, for which HPV infection is associated with nearly all cases. Essential to the oncogenesis of HPV is the function of the viral protein E6 and its role in degrading the cell cycle regulator p53. Degradation of p53, and the resultant loss of cell cycle control, is mediated by E6 recruitment of the E3 ubiquitin ligase E6AP and subsequent ubiquitination of p53. Here, we report the design of a stapled peptide that mimics the LxxLL α-helical domain of E6AP to bind and covalently label a cysteine residue specific to HPV-16 E6. Several acrylamide- and haloacetamide-based warheads were evaluated for reactivity and specificity, and a panel of hydrocarbon-stapled peptides was evaluated for enhanced binding affinity and increased proteolytic stability. Structure-based modeling was used to rationalize the observed trends in the reactivity of the warheads and the impact of the hydrocarbon staple position on the binding affinity of the stapled peptides. The development of a proteolytically stable and reactive peptide represents a new class of peptide-based inhibitors of protein-protein interactions with a potential therapeutic value toward HPV-derived cancers.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"746-757"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934087/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143583757","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}
{"title":"G-Quadruplex-Based Splice Switching as a Therapeutic Approach in Duchenne Muscular Dystrophy.","authors":"Ryo Iwase, Taro Ishiguro, Rintaro I Hara, Tetsuya Nagata, Takanori Yokota","doi":"10.1021/acschembio.4c00805","DOIUrl":"10.1021/acschembio.4c00805","url":null,"abstract":"<p><p>RNA guanine (G)-quadruplexes (rG4) are unique noncanonical structures composed of stacked guanine quadruplexes that play diverse roles in regulating gene expression, from transcription to protein synthesis. This study proposes a new splice-switching therapy using G-quadruplex-inducing antisense oligonucleotides (G-ASOs) to reinstate dystrophin expression in Duchenne muscular dystrophy (DMD) models. G-ASOs consist of two functionally independent domains that enable the formation of RNA/DNA hetero-G-quadruplex (hG4) structures. The antisense domain binds to complementary sequences within the target RNA, while the G-rich domain, which contains a sequence of continuous guanines (G-tract), interacts with the G-rich region of target RNA to form an hG4 structure. This precise binding forms an hG4 structure that effectively interrupts alternative splicing. In contrast to the traditional methods that block sterically, this technique employs steric hindrance by forming hG4 structures. Significantly, our findings show that hG4 structures can still form effectively even when the G-rich region of the target RNA and the antisense sequence are as much as 70 nucleotides apart. To address the challenges associated with G-quadruplex formation via G-ASO self-assembly, we developed bulge-containing G-ASOs. This enhancement improves both the efficiency of hG4 formation and the induction of exon-skipping therapy. In summary, this study highlights the potential of G-ASOs in gene therapy, specifically DMD, and marks significant progress in the development of novel therapeutic strategies. These findings highlight the effectiveness of G-ASOs in exon-skipping therapy and demonstrate the advancements in RNA structural manipulation.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"670-679"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934080/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143539469","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-03-13DOI: 10.1021/acschembio.4c00803
Andrew C D Lemmex, Jeremy Allred, Jason Ostergard, Jake Trask, Hannah N Bui, Michael J M Anderson, Benjamin Kopp, Oakley Streeter, Adam T Smiley, Natalia S Babilonia-Díaz, Bruce R Blazar, LeeAnn Higgins, Peter M Gordon, Joseph M Muretta, Wendy R Gordon
{"title":"Single-Chain Nanobody Inhibition of Notch and Avidity Enhancement Utilizing the β-Pore-Forming Toxin Aerolysin.","authors":"Andrew C D Lemmex, Jeremy Allred, Jason Ostergard, Jake Trask, Hannah N Bui, Michael J M Anderson, Benjamin Kopp, Oakley Streeter, Adam T Smiley, Natalia S Babilonia-Díaz, Bruce R Blazar, LeeAnn Higgins, Peter M Gordon, Joseph M Muretta, Wendy R Gordon","doi":"10.1021/acschembio.4c00803","DOIUrl":"10.1021/acschembio.4c00803","url":null,"abstract":"<p><p>Notch plays critical roles in developmental processes and disease pathogenesis, which have led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling derived from a synthetic phage-display library targeting the Notch negative regulatory region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay with an IC<sub>50</sub> of about 5 μM and in a Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19 μM affinity for the Notch NRR. We addressed the low affinity by fusion to a mutant varient of the β-pore-forming toxin aerolysin, resulting in a significantly improved IC<sub>50</sub> for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with efficacy similar to that of other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"656-669"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143622814","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}
Jessica L Fuller, Ke Shi, Steffen Pockes, Barry C Finzel, Karen H Ashe, Michael A Walters
{"title":"Reengineering of Circularly Permuted Caspase-2 to Enhance Enzyme Stability and Enable Crystallographic Studies.","authors":"Jessica L Fuller, Ke Shi, Steffen Pockes, Barry C Finzel, Karen H Ashe, Michael A Walters","doi":"10.1021/acschembio.4c00795","DOIUrl":"https://doi.org/10.1021/acschembio.4c00795","url":null,"abstract":"<p><p>Caspase activation has been linked to several diseases, including cancer, neurodegeneration, and inflammatory conditions, generating interest in targeting this family of proteases for drug development. Caspase-2 (Casp2) in particular has been implicated in Alzheimer's Disease (AD) by cleaving tau protein into fragment Δtau314, which reversibly impairs cognitive and synaptic function. Thus, Casp2 inhibition could be a useful strategy for therapeutic treatment of AD. To that end, we have previously synthesized and characterized various series of peptide and peptidomimetic inhibitors that demonstrate potency and selectivity for Casp2 over caspase-3 (Casp3). Despite promising developments in the design of selective Casp2 inhibitors, low expression yields of Casp2 hinder crystallographic experiments and make structure-based design challenging. The design of circularly permuted (cp) Casp2 increased protein yields considerably; however, this protein could not be crystallized. This article describes the characterization of ten novel cpCasp2 mutants, designed with the goal of increasing stability and facilitating crystallization. Gratifyingly, engineered mutant JF1cpCasp2 displayed high relative stability and was readily crystallizable with the canonical Casp2 inhibitor AcVDVAD-CHO, leading to what we believe to be the first crystal structures of any reverse caspase in the PDB. Moreover, we have reported the structure of JF1cpCasp2 with our recently described Casp2-selective inhibitor MUR-65, which revealed a unique interaction with Arg417 in the binding pocket. Overall, JF1cpCasp2 has proven valuable for structure-based design and expanding understanding of Casp2 inhibition, with potential implications for drug discovery and the development of more selective compounds.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143672887","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-02-19DOI: 10.1021/acschembio.4c00807
Gerlof P Bosman, Inèz D Stoof, Hans P Bastiaansen, Linda Quarles van Ufford, Justyna M Dobruchowska, Jan-Willem H Langenbach, Bhargavi M Boruah, Kelley W Moremen, Arthur E H Bentlage, Suzanne N Lissenberg-Thunnissen, Gestur Vidarsson, Geert-Jan Boons
{"title":"Bisecting <i>N</i>-Acetylglucosamine of the <i>N</i>-Glycan of Immunoglobulin G Does Not Affect Binding to Fc Gamma Receptors.","authors":"Gerlof P Bosman, Inèz D Stoof, Hans P Bastiaansen, Linda Quarles van Ufford, Justyna M Dobruchowska, Jan-Willem H Langenbach, Bhargavi M Boruah, Kelley W Moremen, Arthur E H Bentlage, Suzanne N Lissenberg-Thunnissen, Gestur Vidarsson, Geert-Jan Boons","doi":"10.1021/acschembio.4c00807","DOIUrl":"10.1021/acschembio.4c00807","url":null,"abstract":"<p><p>Monoclonal antibodies (mAb) produced in 1,4-mannosyl-glycoprotein 4-<i>N</i>-acetylglucosaminyltransferase (MGAT3) overexpressing cell lines have superior <i>in vitro</i> and <i>in vivo</i> activities. The <i>N</i>-glycan of the Fc-region of these mAbs have increased levels of bisecting <i>N</i>-acetylglucosamine (GlcNAc) and reduced core-fucosylation. Although a reduction in core-fucosylation will improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity, the influence of bisecting GlcNAc on these activities has been difficult to probe. Here, we describe the preparation of a unique series of homogeneous glycoforms of trastuzumab (Herceptin) with and without core-fucose and with and without bisecting GlcNAc and examine binding to a comprehensive panel of Fcγ receptors. The glycoforms of trastuzumab were prepared by treatment with wild-type Endo-S2 to cleave the chitobiose core of the <i>N</i>-glycan to leave GlcNAc-Fuc that was exposed to an α-fucosidase to provide trastuzumab-GlcNAc. Glycan oxazolines with and without bisecting GlcNAc were prepared by enzymatic remodeling of a sialoglycopeptide isolated from egg yolk powder, which were employed in transglycosylations with trastuzumab-GlcNAc and trastuzumab-GlcNAc-Fuc catalyzed by Endo-S2 D184M resulting in well-defined glycoforms. As expected, core-fucosylation had a major effect on FcγRIIIa binding, which was not influenced by the presence of bisecting GlcNAc. It was found that an A2-glycan (GlcNAc<sub>2</sub>Man<sub>3</sub>GlcNAc<sub>2</sub>) modified by bisecting GlcNAc cannot be core-fucosylated by FUT8. Thus, bisecting GlcNAc has only an indirect influence on FcγRIIIa binding and subsequent ADCC activity by inhibiting core-fucosylation. The results described here provide an understanding of the properties of therapeutic monoclonal antibodies.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"680-689"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934091/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143456256","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}
ACS Chemical BiologyPub Date : 2025-03-21Epub Date: 2025-03-07DOI: 10.1021/acschembio.4c00783
Gwen Tjallinks, Nicolò Angeleri, Quoc-Thai Nguyen, Barbara Mannucci, Mark Arentshorst, Jaap Visser, Arthur F J Ram, Marco W Fraaije, Andrea Mattevi
{"title":"Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis.","authors":"Gwen Tjallinks, Nicolò Angeleri, Quoc-Thai Nguyen, Barbara Mannucci, Mark Arentshorst, Jaap Visser, Arthur F J Ram, Marco W Fraaije, Andrea Mattevi","doi":"10.1021/acschembio.4c00783","DOIUrl":"10.1021/acschembio.4c00783","url":null,"abstract":"<p><p>Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from <i>Penicillium chrysogenum</i> with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its <i>out</i> state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from <i>P. chrysogenum</i> in <i>Aspergillus niger</i> with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":"646-655"},"PeriodicalIF":3.5,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11934079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571542","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}