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Journal of the American Chemical Society Jan 2014Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have...
Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (βα)8-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (βα)8-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago.
Topics: Aminohydrolases; Archaea; Crystallography, X-Ray; Evolution, Molecular; Extinction, Biological; Models, Molecular; Multienzyme Complexes; Protein Folding; Protein Structure, Secondary
PubMed: 24364418
DOI: 10.1021/ja4115677 -
Molecular Microbiology Mar 2007The assembly of proteins that display complementary activities into supramolecular intra- and extracellular complexes is central to cellular function. One such... (Review)
Review
The assembly of proteins that display complementary activities into supramolecular intra- and extracellular complexes is central to cellular function. One such nanomachine of considerable biological and industrial significance is the plant cell wall degrading apparatus of anaerobic bacteria termed the cellulosome. The Clostridium thermocellum cellulosome assembles through the interaction of a type I dockerin module in the catalytic entities with one of several type I cohesin modules in the non-catalytic scaffolding protein. Recent structural studies have provided the molecular details of how dockerin-cohesin interactions mediate both cellulosome assembly and the retention of the protein complex on the bacterial cell surface. The type I dockerin, which displays near-perfect sequence and structural symmetry, interacts with its cohesin partner through a dual binding mode in which either the N- or C-terminal helix dominate heterodimer formation. The biological significance of this dual binding mode is discussed with respect to the plasticity of the orientation of the catalytic subunits within this supramolecular assembly. The flexibility in the quaternary structure of the cellulosome may reflect the challenges presented by the degradation of a heterogenous recalcitrant insoluble substrate by an intricate macromolecular complex, in which the essential synergy between the catalytic subunits is a key feature of cellulosome function.
Topics: Bacterial Proteins; Cellulase; Clostridium; Membrane Proteins; Multienzyme Complexes; Nanotechnology; Protein Structure, Quaternary; Protein Structure, Tertiary
PubMed: 17367380
DOI: 10.1111/j.1365-2958.2007.05640.x -
Nature Chemical Biology Oct 2011G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both healthy...
G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both healthy and cancerous cells. However, little is known about the response of cytosolic metabolic pathways to GPCR-mediated signaling. Here we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. Through a library screen of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gα(i)-coupled receptors correlates with purinosome assembly and disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling and of the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly and disassembly may be one of the downstream events of mitogenic GPCR signaling in human cancer cells.
Topics: HeLa Cells; Humans; Ligands; Multienzyme Complexes; Protein Multimerization; Purines; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 22020552
DOI: 10.1038/nchembio.690 -
Cell Chemical Biology Jun 2016Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in...
Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in whether primordial enzyme complexes from extinct species displayed a similar degree of functional sophistication. To this end, we used ancestral sequence reconstruction to resurrect the α and β subunits of the tryptophan synthase (TS) complex from the last bacterial common ancestor (LBCA), which presumably existed more than 3.4 billion years ago. We show that the LBCA TS subunits are thermostable and exhibit high catalytic activity. Moreover, they form a complex with αββα stoichiometry whose crystal structure is similar to that of modern TS. Kinetic analysis revealed that the reaction intermediate indole is channeled from the α to the β subunits and suggests that allosteric communication already occurred in LBCA TS.
Topics: Multienzyme Complexes; Salmonella typhimurium; Tryptophan Synthase
PubMed: 27291401
DOI: 10.1016/j.chembiol.2016.05.009 -
The Biochemical Journal Apr 1993
Review
Topics: Amino Acid Sequence; Animals; Cell Nucleus; Cysteine Endopeptidases; Cytoplasm; Molecular Sequence Data; Multienzyme Complexes; Peptides; Proteasome Endopeptidase Complex; RNA
PubMed: 7682410
DOI: 10.1042/bj2910001 -
Nature Structural & Molecular Biology Sep 2019Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity...
Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.
Topics: Catalytic Domain; Chaetomium; Cryoelectron Microscopy; Fungal Proteins; Multienzyme Complexes; Protein Conformation; RNA Precursors
PubMed: 31488907
DOI: 10.1038/s41594-019-0289-8 -
Cell Apr 2012
Topics: Animals; Histone-Lysine N-Methyltransferase; Humans; Multienzyme Complexes; Plants; Yeasts
PubMed: 22500810
DOI: 10.1016/j.cell.2012.03.025 -
Protein Science : a Publication of the... May 2014
Topics: Awards and Prizes; Drug Discovery; Enzyme Inhibitors; Humans; Multienzyme Complexes; Peptides; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase; Toxoplasma; Toxoplasmosis
PubMed: 24692246
DOI: 10.1002/pro.2447 -
The Journal of Biological Chemistry Mar 1994The 26 S proteolytic complex ("26 S proteasome") is a macromolecular assembly thought to be involved in ATP- and ubiquitin-dependent protein degradation in the cytoplasm...
The 26 S proteolytic complex ("26 S proteasome") is a macromolecular assembly thought to be involved in ATP- and ubiquitin-dependent protein degradation in the cytoplasm of higher eukaryotic cells. This complex is composed of one 20 S cylinder particle (multicatalytic proteinase, 20 S proteasome) and two cap-shaped 19 S particles comprising a set of polypeptides in the M(r) range of 35,000-110,000. Here we show that cell supernatant fractions contain both these two subunit complexes as distinct particles as well as assembled to 26 S proteasomes. We have separated and purified all three forms from Xenopus laevis oocytes and have determined their peptidase and protease activities. Using various antibodies specific for either a constitutive p52 polypeptide of the 19 S cap complex or for proteins of the 20 S cylinder particle, we have immunolocalized these complexes in both the cytoplasm and the nucleus of diverse species and cell types. The occurrence of all three forms, the 26 S proteasome, the 20 S cylinder particle, and the 19 S cap complex in the nucleoplasm has also been demonstrated in analyses of isolated giant nuclei from Xenopus oocytes. In addition, we show that the 19 S and 20 S subcomplexes can be released from 26 S proteasomes by ATP depletion and that readdition of ATP to 19 S and 20 S particles in cell extracts leads to the reformation of 26 S proteasomes. We discuss that all three particles (19 S, 20 S, and 26 S) exist in a dynamic equilibrium in both cell compartments and serve cytoplasmic as well as nucleus-specific functions.
Topics: Adenosine Triphosphate; Animals; Cell Nucleus; Chromatography, Ion Exchange; Cysteine Endopeptidases; Cytoplasm; Electrophoresis, Polyacrylamide Gel; Microscopy, Fluorescence; Multienzyme Complexes; Oocytes; Proteasome Endopeptidase Complex; Xenopus laevis
PubMed: 8125997
DOI: No ID Found -
Journal of the American Chemical Society Jun 2009LovF is a highly reducing polyketide synthase (HR-PKS) from the filamentous fungus Aspergillus terreus. LovF synthesizes the alpha-S-methylbutyrate side chain that is...
LovF is a highly reducing polyketide synthase (HR-PKS) from the filamentous fungus Aspergillus terreus. LovF synthesizes the alpha-S-methylbutyrate side chain that is subsequently transferred to monacolin J to yield the cholesterol-lowering natural product lovastatin. In the report, we expressed the full length LovF and reconstituted the megasynthase activities in vitro. We confirmed the diketide product of LovF is offloaded from the LovF ACP domain by the dissociated acyltransferase LovD. This represents the first example of acyltransferase-mediated release of polyketide products from fungal PKSs. We determined LovD primarily interacts with the ACP domain of LovF and the protein-protein interactions lead to highly efficient transfer of the diketide product. The catalytic efficiency is enhanced nearly 1 x 10(6)-fold when LovF was used as the acyl carrier instead of N-acetylcysteamine. Reconstitution and characterization of the LovF offloading mechanism provide new insights into the functions of fungal HR-PKS.
Topics: Acyltransferases; Aspergillus; Fungal Proteins; Multienzyme Complexes; Polyketide Synthases; Recombinant Proteins
PubMed: 19530726
DOI: 10.1021/ja903203g