Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• alosterická regulace MeSH
• bakteriální proteiny metabolismus   chemie   genetika   MeSH
• elektronová kryomikroskopie MeSH
• guanosinpentafosfát metabolismus   MeSH
• guanosintrifosfát * metabolismus   MeSH
• IMP-dehydrogenasa * metabolismus   chemie   antagonisté a inhibitory   MeSH
• inosinmonofosfát metabolismus   chemie   MeSH
• katalytická doména MeSH
• molekulární modely MeSH
• Mycobacterium smegmatis enzymologie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• bakteriální proteiny MeSH
• guanosinpentafosfát MeSH
• guanosintrifosfát * MeSH
• IMP-dehydrogenasa * MeSH
• inosinmonofosfát MeSH

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi-permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non-native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo-EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi-layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.

• Klíčová slova
• GRM2, Klebsiella pneumoniae, bacterial microcompartments, cryo-EM,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• elektronová kryomikroskopie MeSH
• Klebsiella pneumoniae chemie   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• cholin metabolismus   MeSH
• elektronová kryomikroskopie MeSH
• genetické lokusy MeSH
• Klebsiella pneumoniae cytologie   enzymologie   genetika   ultrastruktura   MeSH
• lyasy genetika   metabolismus   MeSH
• organely enzymologie   ultrastruktura   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• syntetická biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cholin MeSH
• lyasy MeSH
• rekombinantní proteiny MeSH