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Cited by 14Microbial Natural Products and BiosynthesisPlant biochemistry and biosynthesisEnzyme Structure and Function

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The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell

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Andreas, Michael P., Giessen, Tobias W.·2024·Nature Communications
DOI 10.1038/s41467-024-54175-4PMC PMC11550324

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Abstract

Abstract Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially responsible for the earthy smell of soil and musty taste of contaminated water. Many bacterial 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in the model soil bacterium Streptomyces griseus . Here, we revise the function of EshA, now referred to as Sg Enc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find that Sg Enc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein. Sg Enc contains a cyclic adenosine monophosphate (cAMP) binding domain (CBD)-fold insertion and a unique metal-binding domain, both displayed on the shell exterior. We show that Sg Enc CBDs do not bind cAMP. We find that 2-MIBS cargo loading is mediated by an N-terminal disordered cargo-loading domain and that 2-MIBS activity and Sg Enc shell structure are not modulated by cAMP. Our work redefines the function of EshA and establishes Family 2B encapsulins as cargo-loaded protein nanocompartments involved in secondary metabolite biosynthesis.

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Fig. 1 2-methylisoborneol biosynthetic gene clusters encoding a Family 2B encapsulin shell protein and its 2-MIBS enzyme cargo are widespread in actinobacteria. a The 2-methylisoborneol (2-MIB) biosynthetic gene cluster from Streptomyces griseus (Sg). The operon encodes a Family 2B encapsulin shell protein (EshA/Enc), 2-methylisoborneol synthase (2-MIBS), and a methyltransferase (MT). kb kilobases. b Reaction scheme for the biosynthesis of 2-MIB. Geranyl pyrophosphate (GPP) is methylated by the MT, yielding 2-methyl GPP, followed by 2-methyl GPP cyclization catalyzed by 2-MIBS resulting in 2-MIB formation. SAM S-adenosyl methionine, SAH S-adenosyl homocysteine, PPi pyrophosphate. c Phylogenetic distribution and encapsulin prevalence in actinobacterial 2-MIB biosynthetic gene clusters (top). Phylogenetic analysis of 723 2-MIBSs found in actinobacterial 2-MIB biosynthetic operons (bottom). Branch numbers (1 to 3) are shown. Nodes are colored by bacterial genus. d SDS-PAGE analysis of purified cargo-loaded Sg Enc shells resulting from heterologous expression in Streptomyces coelicolor A3(2). Source data are provided as a Source Data file. This experiment was repeated independently four times. e Negative stain transmission electron microscopy (TEM) images of purified 2-MIBS-loaded Sg Enc. This experiment was repeated independently four times.
Fig. 1 2-methylisoborneol biosynthetic gene clusters encoding a Family 2B encapsulin shell protein and its 2-MIBS enzyme cargo are widespread in actinobacteria. a The 2-methylisoborneol (2-MIB) biosynthetic gene cluster from Streptomyces griseus (Sg). The operon encodes a Family 2B encapsulin shell protein (EshA/Enc), 2-methylisoborneol synthase (2-MIBS), and a methyltransferase (MT). kb kilobases. b Reaction scheme for the biosynthesis of 2-MIB. Geranyl pyrophosphate (GPP) is methylated by the MT, yielding 2-methyl GPP, followed by 2-methyl GPP cyclization catalyzed by 2-MIBS resulting in 2-MIB formation. SAM S-adenosyl methionine, SAH S-adenosyl homocysteine, PPi pyrophosphate. c Phylogenetic distribution and encapsulin prevalence in actinobacterial 2-MIB biosynthetic gene clusters (top). Phylogenetic analysis of 723 2-MIBSs found in actinobacterial 2-MIB biosynthetic operons (bottom). Branch numbers (1 to 3) are shown. Nodes are colored by bacterial genus. d SDS-PAGE analysis of purified cargo-loaded Sg Enc shells resulting from heterologous expression in Streptomyces coelicolor A3(2). Source data are provided as a Source Data file. This experiment was repeated independently four times. e Negative stain transmission electron microscopy (TEM) images of purified 2-MIBS-loaded Sg Enc. This experiment was repeated independently four times.CC BY-NC-ND · PubMed Central
Fig. 2 Cryo-EM analysis of 2-MIBS-loaded Sg Enc. a Cryo-EM map of the cargo-loaded Sg Enc shell viewed from the exterior down the two-fold axis of symmetry (left). Cut-away view shown on the right. Density corresponding to the HK97-like shell-forming domain is shown in blue. Externally displayed CBDs are shown in green. Internalized 2-MIBS CLD densities shown in yellow. b Exterior view of a Sg Enc pentamer down the five-fold axis of symmetry highlighting the five-fold pore. c Exterior view of a Sg Enc trimer down the three-fold axis of symmetry. d Exterior (left) and interior (right) view down the two-fold axis of symmetry highlighting the large, elongated two-fold pore. When viewed from the exterior, the CBDs partially occlude the two-fold pore. e Structural model of the Sg Enc shell protomer with characteristic HK97-like domain elements and the CBD highlighted with different colors. MBD: metal-binding domain. f 2D class averages of shell-subtracted 2-MIBS-loaded Sg Enc particles (top). 2D class averages of shell-subtracted empty Sg Enc particles (bottom). While discrete internalized cargo puncta are visible for the cargo-loaded sample, no puncta can be seen in empty shells.
Fig. 2 Cryo-EM analysis of 2-MIBS-loaded Sg Enc. a Cryo-EM map of the cargo-loaded Sg Enc shell viewed from the exterior down the two-fold axis of symmetry (left). Cut-away view shown on the right. Density corresponding to the HK97-like shell-forming domain is shown in blue. Externally displayed CBDs are shown in green. Internalized 2-MIBS CLD densities shown in yellow. b Exterior view of a Sg Enc pentamer down the five-fold axis of symmetry highlighting the five-fold pore. c Exterior view of a Sg Enc trimer down the three-fold axis of symmetry. d Exterior (left) and interior (right) view down the two-fold axis of symmetry highlighting the large, elongated two-fold pore. When viewed from the exterior, the CBDs partially occlude the two-fold pore. e Structural model of the Sg Enc shell protomer with characteristic HK97-like domain elements and the CBD highlighted with different colors. MBD: metal-binding domain. f 2D class averages of shell-subtracted 2-MIBS-loaded Sg Enc particles (top). 2D class averages of shell-subtracted empty Sg Enc particles (bottom). While discrete internalized cargo puncta are visible for the cargo-loaded sample, no puncta can be seen in empty shells.CC BY-NC-ND · PubMed Central
Fig. 3 Structural analysis of the Sg Enc CBD. a An overview of the structure of the Sg Enc CBD. The metal-binding subdomain (MDB) is shown in purple and the remainder of the CBD is shown in green and light grey. b The E. coli (Ec) CAP CBD (residues 1 to 116) bound to cAMP (PDB ID: 1G6N)31 colored analogously to a. cAMP is shown as yellow sticks. c Surface representations of the Sg Enc CBD binding pocket colored according to hydrophobicity (left) and surface charge (right). d Surface representations of the Ec CAP CBD binding pocket colored according to hydrophobicity (left) and surface charge (right). Bound cAMP is shown as yellow sticks. e Key Sg Enc CBD residues forming the CBD binding pocket. Individual residues are colored by conservation as predicted by ConSurf analysis110–112. ConSurf conservation score key shown in h. Source data are provided as a Source Data file. f Key Ec CAP CBD residues in the CBD binding pocket involved in cAMP binding. Individual residues are colored by conservation as predicted by ConSurf analysis. g The CBD interacts with the HK97-domain of the Sg Enc protomer predominantly via electrostatic interactions mediated by the MBD. The interaction interfaces are outlined on the HK97-domain surface (left) for the CBD (green) and the MBD (purple). Conversely, interaction interfaces of the HK97-domain with the CBD and MBD are outlined in blue (right). h The MBD is a highly conserved feature of Family 2B encapsulin shell proteins. Side chains and backbone atoms coordinating the metal ion are colored according to sequence conservation as predicted by ConSurf analysis. Extracted cryo-EM metal ion density is shown in green.
Fig. 3 Structural analysis of the Sg Enc CBD. a An overview of the structure of the Sg Enc CBD. The metal-binding subdomain (MDB) is shown in purple and the remainder of the CBD is shown in green and light grey. b The E. coli (Ec) CAP CBD (residues 1 to 116) bound to cAMP (PDB ID: 1G6N)31 colored analogously to a. cAMP is shown as yellow sticks. c Surface representations of the Sg Enc CBD binding pocket colored according to hydrophobicity (left) and surface charge (right). d Surface representations of the Ec CAP CBD binding pocket colored according to hydrophobicity (left) and surface charge (right). Bound cAMP is shown as yellow sticks. e Key Sg Enc CBD residues forming the CBD binding pocket. Individual residues are colored by conservation as predicted by ConSurf analysis110–112. ConSurf conservation score key shown in h. Source data are provided as a Source Data file. f Key Ec CAP CBD residues in the CBD binding pocket involved in cAMP binding. Individual residues are colored by conservation as predicted by ConSurf analysis. g The CBD interacts with the HK97-domain of the Sg Enc protomer predominantly via electrostatic interactions mediated by the MBD. The interaction interfaces are outlined on the HK97-domain surface (left) for the CBD (green) and the MBD (purple). Conversely, interaction interfaces of the HK97-domain with the CBD and MBD are outlined in blue (right). h The MBD is a highly conserved feature of Family 2B encapsulin shell proteins. Side chains and backbone atoms coordinating the metal ion are colored according to sequence conservation as predicted by ConSurf analysis. Extracted cryo-EM metal ion density is shown in green.CC BY-NC-ND · PubMed Central
Fig. 4 CLD-mediated cargo-loading of Sg Enc. a Cut-way view of an Sg Enc shell model shown in electrostatic surface representation with extracted cryo-EM CLD densities obtained via asymmetric C1 refinement shown in yellow (left). Zoom-in highlighting CLD density bound to the interior of the shell at the three-fold axis of symmetry (right). b The interior of Sg Enc surrounding the three-fold axis of symmetry shows strong sequence conservation throughout Family 2B encapsulins. Residues are colored based on ConSurf conservation analysis. The three protomers at the three-fold symmetry axis are highlighted by differently colored backgrounds. Conserved hydrophobic residues F430, Y441, L285, and L264 are shown as stick models and are located directly below the observed CLD density (transparent yellow). c AlphaFold model of Sg 2-MIBS highlighting predicted secondary structure elements within the disordered N-terminal CLD. Five CLD elements – CLD_A to CLD_E – are shown in different colors. The structured TC domain of Sg 2-MIBS is shown in surface representation (purple). d Sg 2-MIB CLD sequence colored by ConSurf conservation score. A sequence logo for the conserved motif within CLD_D is shown. Source data are provided as a Source Data file. e SDS-PAGE analysis of purified Sg Enc shells. Deletion of the CLD abolishes cargo loading of Sg 2-MIBS when co-expressed with Sg Enc. Sg Enc_2-MIBS: 2-MIBS-loaded Sg Enc. ΔCLD: truncation mutant missing all of the CLD. This experiment was repeated independently three times. Source data are provided as a Source Data file. f Systematic deletion of the five identified CLD regions – CLD_A to CLD_E (left). A co-expression system of Sg Enc and CLD-fused mNeonGreen (mNeon), employed as a reporter, was used. Native PAGE analysis of purified truncation mutants shown on the right. Coomassie protein stain (right, top) and mNeonGreen fluorescence (right, middle) are shown. Normalized loading percentages based on gel densitometry and fluorescence analysis are shown (right, bottom). Data are shown as mean values, with error bars representing the standard deviation of four independent experiments. Source data are provided as a Source Data file.
Fig. 4 CLD-mediated cargo-loading of Sg Enc. a Cut-way view of an Sg Enc shell model shown in electrostatic surface representation with extracted cryo-EM CLD densities obtained via asymmetric C1 refinement shown in yellow (left). Zoom-in highlighting CLD density bound to the interior of the shell at the three-fold axis of symmetry (right). b The interior of Sg Enc surrounding the three-fold axis of symmetry shows strong sequence conservation throughout Family 2B encapsulins. Residues are colored based on ConSurf conservation analysis. The three protomers at the three-fold symmetry axis are highlighted by differently colored backgrounds. Conserved hydrophobic residues F430, Y441, L285, and L264 are shown as stick models and are located directly below the observed CLD density (transparent yellow). c AlphaFold model of Sg 2-MIBS highlighting predicted secondary structure elements within the disordered N-terminal CLD. Five CLD elements – CLD_A to CLD_E – are shown in different colors. The structured TC domain of Sg 2-MIBS is shown in surface representation (purple). d Sg 2-MIB CLD sequence colored by ConSurf conservation score. A sequence logo for the conserved motif within CLD_D is shown. Source data are provided as a Source Data file. e SDS-PAGE analysis of purified Sg Enc shells. Deletion of the CLD abolishes cargo loading of Sg 2-MIBS when co-expressed with Sg Enc. Sg Enc_2-MIBS: 2-MIBS-loaded Sg Enc. ΔCLD: truncation mutant missing all of the CLD. This experiment was repeated independently three times. Source data are provided as a Source Data file. f Systematic deletion of the five identified CLD regions – CLD_A to CLD_E (left). A co-expression system of Sg Enc and CLD-fused mNeonGreen (mNeon), employed as a reporter, was used. Native PAGE analysis of purified truncation mutants shown on the right. Coomassie protein stain (right, top) and mNeonGreen fluorescence (right, middle) are shown. Normalized loading percentages based on gel densitometry and fluorescence analysis are shown (right, bottom). Data are shown as mean values, with error bars representing the standard deviation of four independent experiments. Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 5 The effect of cAMP on Sg 2-MIBS activity and cAMP binding to Sg Enc CBD. a In vitro activity assays of 2-MIBS-loaded Sg Enc in the absence and presence of cAMP. Free 2-MIBS activity is shown for reference. kcat/Km: catalytic efficiency of 2-MIBS. Data are shown as mean values, with error bars representing the standard deviation of three independent experiments. Source data are provided as a Source Data file. b GC-MS analysis of 2-MIBS activity assays. All assays yielded 2-MIB as the main product, with its diagnostic mass fragment (m/z + 95) identified as the most abundant ion14. A 2-MIB standard is shown for reference (top). Source data are provided as a Source Data file. c Differential scanning fluorimetry (DSF) thermal melts of 2-MIBS-loaded Sg Enc in the absence or presence of cAMP. Tm: melting temperature. Source data are provided as a Source Data file. d 2-MIBS-loaded Sg Enc binding to cAMP-modified agarose resin. A representative SDS-PAGE gel is shown demonstrating that 2-MIBS-loaded Sg Enc is unable to bind to cAMP-modified agarose resin. The positive control Ec CAP is able to bind to the resin. Load: sample loaded onto the resin. 20 mM cAMP (orange): elution from the resin with 20 mM cAMP. This experiment was repeated independently four times. Source data are provided as a Source Data file. e Cryo-EM analysis of Sg Enc in the presence of cAMP. No significant conformational changes of the CBDs or the HK97-domain could be observed when compared to cryo-EM maps obtained in the absence of cAMP. Improved CBD density was obtained via 3D classification followed C1 homogeneous refinement, but no conformational changes could be observed. f Cryo-EM density of the β-barrel CBD binding site is shown (top). No additional density could be observed in the presence of cAMP. The metal-binding subdomain (MBD) is shown in purple. Structural model of the binding site with binding site residues shown as stick (bottom).
Fig. 5 The effect of cAMP on Sg 2-MIBS activity and cAMP binding to Sg Enc CBD. a In vitro activity assays of 2-MIBS-loaded Sg Enc in the absence and presence of cAMP. Free 2-MIBS activity is shown for reference. kcat/Km: catalytic efficiency of 2-MIBS. Data are shown as mean values, with error bars representing the standard deviation of three independent experiments. Source data are provided as a Source Data file. b GC-MS analysis of 2-MIBS activity assays. All assays yielded 2-MIB as the main product, with its diagnostic mass fragment (m/z + 95) identified as the most abundant ion14. A 2-MIB standard is shown for reference (top). Source data are provided as a Source Data file. c Differential scanning fluorimetry (DSF) thermal melts of 2-MIBS-loaded Sg Enc in the absence or presence of cAMP. Tm: melting temperature. Source data are provided as a Source Data file. d 2-MIBS-loaded Sg Enc binding to cAMP-modified agarose resin. A representative SDS-PAGE gel is shown demonstrating that 2-MIBS-loaded Sg Enc is unable to bind to cAMP-modified agarose resin. The positive control Ec CAP is able to bind to the resin. Load: sample loaded onto the resin. 20 mM cAMP (orange): elution from the resin with 20 mM cAMP. This experiment was repeated independently four times. Source data are provided as a Source Data file. e Cryo-EM analysis of Sg Enc in the presence of cAMP. No significant conformational changes of the CBDs or the HK97-domain could be observed when compared to cryo-EM maps obtained in the absence of cAMP. Improved CBD density was obtained via 3D classification followed C1 homogeneous refinement, but no conformational changes could be observed. f Cryo-EM density of the β-barrel CBD binding site is shown (top). No additional density could be observed in the presence of cAMP. The metal-binding subdomain (MBD) is shown in purple. Structural model of the binding site with binding site residues shown as stick (bottom).CC BY-NC-ND · PubMed Central

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  1. Christianson, D. W. Structural and Chemical Biology of Terpenoid Cyclases. Chem. Rev. 117, 11570–11648 (2017). · DOI
  2. Rudolf, J. D., Alsup, T. A., Xu, B. & Li, Z. Bacterial terpenome. Nat. Prod. Rep. 38, 905–980 (2021). · DOI
  3. Tholl, D. in Biotechnology of Isoprenoids (eds Jens Schrader & Jörg Bohlmann) 63-106 (Springer International Publishing, 2015). · DOI
  4. Harvey, A. L. Natural products in drug discovery. Drug Discov. Today 13, 894–901 (2008). · DOI
  5. Cane, D. E. & Ikeda, H. Exploration and mining of the bacterial terpenome. Acc. Chem. Res. 45, 463–472 (2012). · DOI
  6. Dickschat, J. S. Bacterial terpene cyclases. Nat. Prod. Rep. 33, 87–110 (2016). · DOI
  7. Juttner, F. & Watson, S. B. Biochemical and ecological control of geosmin and 2-methylisoborneol in source waters. Appl Environ. Microbiol 73, 4395–4406 (2007). · DOI
  8. Garbeva, P., Avalos, M., Ulanova, D., van Wezel, G. P. & Dickschat, J. S. Volatile sensation: The chemical ecology of the earthy odorant geosmin. Environ. Microbiol 25, 1565–1574 (2023). · DOI
  9. Zaroubi, L. et al. The Ubiquitous Soil Terpene Geosmin Acts as a Warning Chemical. Appl Environ. Microbiol 88, e0009322 (2022). · DOI
  10. Saha, M. & Fink, P. Algal volatiles - the overlooked chemical language of aquatic primary producers. Biol. Rev. Camb. Philos. Soc. 97, 2162–2173 (2022). · DOI
  11. Wang, C. M. & Cane, D. E. Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor. J. Am. Chem. Soc. 130, 8908–8909 (2008). · DOI
  12. Giglio, S., Chou, W. K., Ikeda, H., Cane, D. E. & Monis, P. T. Biosynthesis of 2-methylisoborneol in cyanobacteria. Environ. Sci. Technol. 45, 992–998 (2011). · DOI
  13. Wang, Z., Xu, Y., Shao, J., Wang, J. & Li, R. Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light. PLoS One 6, e18665 (2011). · DOI
  14. Dickschat, J. S. et al. Biosynthesis of the off-flavor 2-methylisoborneol by the myxobacterium Nannocystis exedens. Angew. Chem. Int Ed. Engl. 46, 8287–8290 (2007). · DOI
  15. Avalos, M. et al. Biosynthesis, evolution and ecology of microbial terpenoids. Nat. Prod. Rep. 39, 249–272 (2022). · DOI
  16. Scholler, C. E., Gurtler, H., Pedersen, R., Molin, S. & Wilkins, K. Volatile metabolites from actinomycetes. J. Agric Food Chem. 50, 2615–2621 (2002). · DOI
  17. Citron, C. A., Gleitzmann, J., Laurenzano, G., Pukall, R. & Dickschat, J. S. Terpenoids are widespread in actinomycetes: a correlation of secondary metabolism and genome data. Chembiochem 13, 202–214 (2012). · DOI
  18. Larsen, T. O. & Frisvad, J. C. Characterization of volatile metabolites from 47 Penicillium taxa. Mycological Res. 99, 1153–1166 (1995). · DOI
  19. Wilkins, K. Volatile Organic Metabolites from Selected Streptomyces Strains. Actinomycetologica 23, 27–33 (2009). · DOI
  20. Medsker, L. L., Jenkins, D., Thomas, J. F. & Koch, C. Odorous compounds in natural waters. 2-exo-Hydroxy-2-methylbornane, the major odorous compound produced by several actinomycetes. Environ. Sci. Technol. 3, 476–477 (1969). · DOI
  21. Gerber, N. N. A volatile metabolite of actinomycetes, 2-methylisoborneol. J. Antibiot. (Tokyo) 22, 508–509 (1969). · DOI
  22. Bentley, R. & Meganathan, R. Geosmin and methylisoborneol biosynthesis in streptomycetes. Evidence for an isoprenoid pathway and its absence in non-differentiating isolates. FEBS Lett. 125, 220–222 (1981). · DOI
  23. Gu, B., Liang, L. F. & Dickschat, J. S. Functions of enzyme domains in 2-methylisoborneol biosynthesis and enzymatic synthesis of non-natural analogs. Beilstein J. Org. Chem. 19, 1452–1459 (2023). · DOI
  24. Koksal, M., Chou, W. K., Cane, D. E. & Christianson, D. W. Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate. Biochemistry 51, 3011–3020 (2012). · DOI
  25. Brock, N. L., Ravella, S. R., Schulz, S. & Dickschat, J. S. A detailed view of 2-methylisoborneol biosynthesis. Angew. Chem. Int Ed. Engl. 52, 2100–2104 (2013). · DOI
  26. Gu, B., Hou, A. & Dickschat, J. S. The stereochemical course of 2-methylisoborneol biosynthesis. Beilstein J. Org. Chem. 18, 818–824 (2022). · DOI
  27. Gu, B. & Dickschat, J. S. A non-natural biosynthesis pathway toward 2-methylisoborneol. Chem. Commun. (Camb.) 58, 4316–4319 (2022). · DOI
  28. Komatsu, M., Tsuda, M., Omura, S., Oikawa, H. & Ikeda, H. Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proc. Natl Acad. Sci. USA 105, 7422–7427 (2008). · DOI
  29. Koksal, M., Chou, W. K., Cane, D. E. & Christianson, D. W. Structure of geranyl diphosphate C-methyltransferase from Streptomyces coelicolor and implications for the mechanism of isoprenoid modification. Biochemistry 51, 3003–3010 (2012). · DOI
  30. Kawamoto, S. et al. Molecular and functional analyses of the gene (eshA) encoding the 52-kilodalton protein of Streptomyces coelicolor A3(2) required for antibiotic production. J. Bacteriol. 183, 6009–6016 (2001). · DOI
  31. Passner, J. M., Schultz, S. C. & Steitz, T. A. Modeling the cAMP-induced Allosteric Transition Using the Crystal Structure of CAP-cAMP at 2.1Å Resolution. J. Mol. Biol. 304, 847–859 (2000). · DOI
  32. Andreas, M. P. & Giessen, T. W. Large-scale computational discovery and analysis of virus-derived microbial nanocompartments. Nat. Commun. 12, 4748 (2021). · DOI
  33. Nichols, R. J. et al. Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism. Elife 10. https://doi.org/10.7554/eLife.59288 (2021). · DOI
  34. Giessen, T. W. Encapsulins. Annu Rev Biochem. https://doi.org/10.1146/annurev-biochem-040320-102858 (2022). · DOI
  35. Nichols, R. J., Cassidy-Amstutz, C., Chaijarasphong, T. & Savage, D. F. Encapsulins: molecular biology of the shell. Crit. Rev. Biochem Mol. Biol. 52, 583–594 (2017). · DOI
  36. Sutter, M. et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nat. Struct. Mol. Biol. 15, 939–947 (2008). · DOI
  37. Giessen, T. W. & Silver, P. A. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat. Microbiol 2, 17029 (2017). · DOI
  38. Heinemann, J. et al. Fossil record of an archaeal HK97-like provirus. Virology 417, 362–368 (2011). · DOI
  39. Altenburg, W. J., Rollins, N., Silver, P. A. & Giessen, T. W. Exploring targeting peptide-shell interactions in encapsulin nanocompartments. Sci. Rep. 11, 4951 (2021). · DOI
  40. McHugh, C. A. et al. A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. EMBO J. 33, 1896–1911 (2014). · DOI
  41. Giessen, T. W. et al. Large protein organelles form a new iron sequestration system with high storage capacity. Elife 8. https://doi.org/10.7554/eLife.46070 (2019). · DOI
  42. Akita, F. et al. The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses. J. Mol. Biol. 368, 1469–1483 (2007). · DOI
  43. Kwon, S., Andreas, M. P. & Giessen, T. W. Structure and heterogeneity of a highly cargo-loaded encapsulin shell. J. Struct. Biol. 215, 108022 (2023). · DOI
  44. Eren, E. et al. Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism. Structure 30, 551–563 e554 (2022). · DOI
  45. Jones, J. A., Andreas, M. P. & Giessen, T. W. Structural basis for peroxidase encapsulation inside the encapsulin from the Gram-negative pathogen Klebsiella pneumoniae. Nat. Commun. 15, 2558 (2024). · DOI
  46. Sugano, Y. & Yoshida, T. DyP-Type Peroxidases: Recent Advances and Perspectives. Int J Mol Sci. 22. https://doi.org/10.3390/ijms22115556 (2021). · DOI
  47. Almeida, A. V. et al. Condensation and Protection of DNA by the Myxococcus xanthus Encapsulin: A Novel Function. Int J Mol Sci 23. https://doi.org/10.3390/ijms23147829 (2022). · DOI
  48. Benisch, R., Andreas, M. P. & Giessen, T. W. A widespread bacterial protein compartment sequesters and stores elemental sulfur. Sci. Adv. 10, eadk9345 (2024). · DOI
  49. Kwak, J., McCue, L. A., Trczianka, K. & Kendrick, K. E. Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus. J. Bacteriol. 183, 3004–3015 (2001). · DOI
  50. Saito, N., Matsubara, K., Watanabe, M., Kato, F. & Ochi, K. Genetic and biochemical characterization of EshA, a protein that forms large multimers and affects developmental processes in Streptomyces griseus. J. Biol. Chem. 278, 5902–5911 (2003). · DOI
  51. Saito, N. et al. EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3(2). J. Bacteriol. 188, 4952–4961 (2006). · DOI
  52. Jones, J. A., Benisch, R. & Giessen, T. W. Encapsulin cargo loading: progress and potential. J. Mater. Chem. B 11, 4377–4388 (2023). · DOI
  53. Gu, B. & Dickschat, J. S. Functional Characterisation of Highly Conserved and Structurally Prominent Residues of 2-Methylisoborneol Synthase. Chemistry 29, e202300775 (2023). · DOI
  54. Berman, H. M. et al. The cAMP binding domain: an ancient signaling module. Proc. Natl Acad. Sci. USA 102, 45–50 (2005). · DOI
  55. Kannan, N. et al. Evolution of allostery in the cyclic nucleotide binding module. Genome Biol. 8, R264 (2007). · DOI
  56. Krol, E., Werel, L., Essen, L. O. & Becker, A. Structural and functional diversity of bacterial cyclic nucleotide perception by CRP proteins. microLife 4. https://doi.org/10.1093/femsml/uqad024 (2023). · DOI
  57. Popovych, N., Tzeng, S.-R., Tonelli, M., Ebright, R. H. & Kalodimos, C. G. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein. Proc. Natl Acad. Sci. 106, 6927–6932 (2009). · DOI
  58. Belduz, A. O., Lee, E. J. & Harman, J. G. Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket. Nucleic Acids Res 21, 1827–1835 (1993). · DOI
  59. Gronenborn, A. M., Sandulache, R., Gärtner, S. & Clore, G. M. Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli. Biochem J. 253, 801–807 (1988). · DOI
  60. Moore, J., Kantorow, M., Vanderzwaag, D. & McKenney, K. Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation. J. Bacteriol. 174, 8030–8035 (1992). · DOI
  61. Lanzilotta, W. N. et al. Structure of the CO sensing transcription activator CooA. Nat. Struct. Biol. 7, 876–880 (2000). · DOI
  62. Joyce, M. G. et al. CprK Crystal Structures Reveal Mechanism for Transcriptional Control of Halorespiration *. J. Biol. Chem. 281, 28318–28325 (2006). · DOI
  63. Zhao, M.-X. et al. Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate. Proc. Natl Acad. Sci. 107, 12487–12492 (2010). · DOI
  64. Brelidze, T. I., Carlson, A. E., Sankaran, B. & Zagotta, W. N. Structure of the carboxy-terminal region of a KCNH channel. Nature 481, 530–533 (2012). · DOI
  65. Marques-Carvalho, M. J. et al. Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel. J. Mol. Biol. 423, 34–46 (2012). · DOI
  66. Haitin, Y., Carlson, A. E. & Zagotta, W. N. The structural mechanism of KCNH-channel regulation by the eag domain. Nature 501, 444–448 (2013). · DOI
  67. Holm, L., Laiho, A., Törönen, P. & Salgado, M. DALI shines a light on remote homologs: One hundred discoveries. Protein Sci. 32, e4519 (2023). · DOI
  68. Gabler, F. et al. Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Curr. Protoc. Bioinforma. 72, e108 (2020). · DOI · Paper page
  69. Zimmermann, L. et al. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J. Mol. Biol. 430, 2237–2243 (2018). · DOI · Paper page
  70. Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022). · DOI · Paper page
  71. Cassidy-Amstutz, C. et al. Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry 55, 3461–3468 (2016). · DOI
  72. Jones, J. A., Andreas, M. P. & Giessen, T. W. Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage. Biomacromolecules 24, 1388–1399 (2023). · DOI
  73. Kwon, S. & Giessen, T. W. Engineered Protein Nanocages for Concurrent RNA and Protein Packaging In Vivo. ACS Synth. Biol. 11, 3504–3515 (2022). · DOI
  74. Faylo, J. L. et al. Structural insight on assembly-line catalysis in terpene biosynthesis. Nat. Commun. 12, 3487 (2021). · DOI
  75. Ronnebaum, T. A., Eaton, S. A., Brackhahn, E. A. E. & Christianson, D. W. Engineering the Prenyltransferase Domain of a Bifunctional Assembly-Line Terpene Synthase. Biochemistry 60, 3162–3172 (2021). · DOI
  76. Ronnebaum, T. A., Gupta, K. & Christianson, D. W. Higher-order oligomerization of a chimeric αβγ bifunctional diterpene synthase with prenyltransferase and class II cyclase activities is concentration-dependent. J. Struct. Biol. 210, 107463 (2020). · DOI
  77. Gao, K., Oerlemans, R. & Groves, M. R. Theory and applications of differential scanning fluorimetry in early-stage drug discovery. Biophys. Rev. 12, 85–104 (2020). · DOI
  78. Dremier, S., Kopperud, R., Doskeland, S. O., Dumont, J. E. & Maenhaut, C. Search for new cyclic AMP-binding proteins. FEBS Lett. 546, 103–107 (2003). · DOI
  79. Bertinetti, D. et al. Chemical tools selectively target components of the PKA system. BMC Chem. Biol. 9, 3 (2009). · DOI
  80. Endoh, T. & Engel Joanne, N. CbpA: a Polarly Localized Novel Cyclic AMP-Binding Protein in Pseudomonas aeruginosa. J. Bacteriol. 191, 7193–7205 (2009). · DOI
  81. Jäger, A. V. et al. Identification of novel cyclic nucleotide binding proteins in Trypanosoma cruzi. Mol. Biochemical Parasitol. 198, 104–112 (2014). · DOI
  82. Ross, J. et al. Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment. Sci. Adv. 8, eabj4461 (2022). · DOI
  83. Adamson, L. S. R. et al. Pore structure controls stability and molecular flux in engineered protein cages. Sci. Adv. 8, eabl7346 (2022). · DOI
  84. Joyce, M. G. et al. CprK crystal structures reveal mechanism for transcriptional control of halorespiration. J. Biol. Chem. 281, 28318–28325 (2006). · DOI
  85. Ober, V. T. et al. Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens. eLife 12, RP91040 (2024). · DOI
  86. Bachmaier, S. et al. Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma. Nat. Commun. 10, 1421 (2019). · DOI
  87. Zallot, R., Oberg, N. & Gerlt, J. A. The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 58, 4169–4182 (2019). · DOI
  88. Oberg, N., Zallot, R. & Gerlt, J. A. EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools. J. Mol. Biol. https://doi.org/10.1016/j.jmb.2023.168018 (2023). · DOI
  89. Lemoine, F. et al. NGPhylogeny.fr: new generation phylogenetic services for non-specialists. Nucleic Acids Res. 47, W260–W265 (2019). · DOI
  90. Katoh, K. & Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evolution 30, 772–780 (2013). · DOI · Paper page
  91. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments. PLOS ONE 5, e9490 (2010). · DOI · Paper page
  92. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix. Mol. Biol. Evolution 26, 1641–1650 (2009). · DOI · Paper page
  93. Lemoine, F. et al. Renewing Felsenstein’s phylogenetic bootstrap in the era of big data. Nature 556, 452–456 (2018). · DOI
  94. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021). · DOI
  95. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498–2504 (2003). · DOI · Paper page
  96. Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic acids Res. 50, W276–W279 (2022). · DOI
  97. Andreas, M. P. & Giessen, T. W. Heterologous expression and purification of encapsulins in Streptomyces coelicolor. MethodsX 9, 101787 (2022). · DOI
  98. Andreas, M. P. & Giessen, T. W. Cyclodipeptide oxidase is an enzyme filament. Nat Commun. 15, 3574 (2024). · DOI
  99. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012). · DOI · Paper page
  100. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005). · DOI
  101. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). · DOI · Paper page
  102. UniProt, C. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res 51, D523–D531 (2023). · DOI · Paper page
  103. Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). · DOI
  104. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D: Biol. Crystallogr. 66, 486–501 (2010). · DOI · Paper page
  105. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D. Biol. Crystallogr 60, 2126–2132 (2004). · DOI · Paper page
  106. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D. Struct. Biol. 75, 861–877 (2019). · DOI · Paper page
  107. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D. Biol. Crystallogr 66, 213–221 (2010). · DOI · Paper page
  108. Yariv, B. et al. Using evolutionary data to make sense of macromolecules with a “face-lifted” ConSurf. Protein Sci. 32, e4582 (2023). · DOI
  109. Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016). · DOI · Paper page
  110. Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010). · DOI

Metadata

Authors
Andreas, M. P., & Giessen, T. W.
Affiliations
University of Michigan (US)
Corresponding author
Giessen, Tobias W.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-11-09
Volume
15
Issue
1
Pages
9715-9715
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-54175-4
PMC
PMC11550324
OpenAlex
W4404198673
Cited by
14
References
3
Citations by year
2026: 4 · 2025: 9 · 2024: 1
FWCI
3.98
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nd)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35GM133325)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
2-methylisoborneol, encapsulin, Streptomyces griseus, protein shell, cargo loading, cAMP-binding domain, metal-binding domain, secondary metabolite biosynthesis, cryo-electron microscopy

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This page
CoChat. (2026). Citation check: The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell. https://app.cochat.ai/papers/10.1038/s41467-024-54175-4
The paper
Andreas, M. P., & Giessen, T. W. (2024). The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell. Nature Communications. https://doi.org/10.1038/s41467-024-54175-4