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Current Opinion in Structural Biology Dec 2019The duplication of chromosomal DNA is a key cell cycle event that involves the controlled, bidirectional assembly of the replicative machinery. In a tightly regulated,... (Review)
Review
The duplication of chromosomal DNA is a key cell cycle event that involves the controlled, bidirectional assembly of the replicative machinery. In a tightly regulated, multi-step reaction, replicative helicases and other components of the DNA synthesis apparatus are recruited to replication start sites. Although the molecular approaches for assembling this machinery vary between the different domains of life, a common theme revolves around the use of ATP-dependent initiation factors to recognize and remodel origins and to load replicative helicases in a bidirectional manner onto DNA. This review summarizes recent advances in understanding the mechanisms of replication initiation in eukaryotes, focusing on how the replicative helicase is loaded in this system.
Topics: Adenosine Triphosphate; DNA; DNA Helicases; DNA Replication; DNA-Directed DNA Polymerase; Hydrolysis; Models, Molecular; Multienzyme Complexes; Protein Binding; Quantitative Structure-Activity Relationship; Replication Origin
PubMed: 31630057
DOI: 10.1016/j.sbi.2019.08.007 -
International Journal of Molecular... Aug 2023Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive... (Review)
Review
Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme-inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal enzyme initial velocity versus inhibitor concentration, partial inhibitors produce hyperbolic plots. Similarly, dose-response curves show residual fractional activity of enzymes at high doses. This article reviews the theory and methods of analysis and discusses the significance of this type of reversible enzyme inhibition in metabolic processes, and its implications in pharmacology and toxicology.
Topics: Multienzyme Complexes
PubMed: 37629158
DOI: 10.3390/ijms241612973 -
ACS Chemical Biology May 2021Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and...
Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications.
Topics: Antibodies; Biocompatible Materials; Cell Membrane Permeability; Drug Compounding; Drug Liberation; Humans; Immunoglobulins; Molecular Targeted Therapy; Multienzyme Complexes; Nanocapsules; Protein Engineering; Surface Properties; Tissue Distribution
PubMed: 33881303
DOI: 10.1021/acschembio.1c00007 -
Neurochemical Research Oct 2019Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early... (Review)
Review
Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early death; the highest carrier rate has been found among Ashkenazi Jews. Acute clinical episodes usually involve severe metabolic decompensation and lactate acidosis that result in neurological, cardiological, and/or hepatological manifestations. Clinical severity is due to the fact that LADH is a common E3 subunit to the alpha-ketoglutarate, pyruvate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes, and is also a constituent in the glycine cleavage system, thus a loss in LADH function adversely affects multiple key metabolic routes. However, the severe clinical pictures frequently still do not parallel the LADH activity loss, which implies the involvement of auxiliary biochemical mechanisms; enhanced reactive oxygen species generation as well as affinity loss for multienzyme complexes proved to be key auxiliary exacerbating pathomechanisms. This review provides an overview and an up-to-date molecular insight into the pathomechanisms of this disease in light of the structural conclusions drawn from the first crystal structure of a disease-causing hE3 variant determined recently in our laboratory.
Topics: Acidosis, Lactic; Amino Acid Oxidoreductases; Cytochrome-B(5) Reductase; Dihydrolipoamide Dehydrogenase; Humans; Maple Syrup Urine Disease; Methemoglobinemia; Multienzyme Complexes; Pyruvic Acid; Reactive Oxygen Species; Transferases
PubMed: 30847858
DOI: 10.1007/s11064-019-02766-9 -
Autophagy Sep 2020The AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by sensing the metabolic status of the cell. AMPK is regulated by phosphorylation and...
UNLABELLED
The AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by sensing the metabolic status of the cell. AMPK is regulated by phosphorylation and dephosphorylation as a result of changing AMP/ATP levels and by removal of inhibitory ubiquitin residues by USP10. In this context, we identified the GID-complex, an evolutionarily conserved ubiquitin-ligase-complex (E3), as a negative regulator of AMPK activity. Our data show that the GID-complex targets AMPK for ubiquitination thereby altering its activity. Cells depleted of GID-subunits mimic a state of starvation as shown by increased AMPK activity and macroautophagic/autophagic flux as well as reduced MTOR activation. Consistently, -genes knockdown in results in increased organismal lifespan. This study may contribute to understand metabolic disorders such as type 2 diabetes mellitus and morbid obesity and implements alternative therapeutic approaches to alter AMPK activity.
ABBREVIATIONS
ACTB: actin, beta; ADP: adenosine diphosphate; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ARMC8: armadillo repeat containing 8; ATP: adenosine triphosphate; BafA1: bafilomycin A; BCAA: branched chain amino acid; BICC1: BicC family RNA binding protein 1; BSA: bovine serum albumin; CAMKK2 kinase: calcium/calmodulin dependent protein kinase kinase 2, beta; CHX: cycloheximide; DMEM: Dulbecco's modified Eagle's medium; E1: ubiquitin-activating enzyme; E2: ubiquitin-conjugating enzyme; E3: ubiquitin ligase; ECAR: extracellular acidification rate; FACS: fluorescent associated cell sorter; FBP1: fructose-bisphosphatase 1; FCCP: carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; G6P: glucose-6-phosphate; GDP: guanosine diphosphate; GFP: green fluorescent protein; GID: glucose induced degradation deficient; GMP: guanosine monophosphate; GTP: guanosine triphosphate; HBP1: high mobility group box transcription factor 1; HPRT: hypoxanthine guanine phosphoribosyl transferase; KO: knock out; LE: long exposure; MAEA: macrophage erythroblast attacher; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MKLN1: muskelin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin; NES: normalized enrichment score; OCR: oxygen consumption rate; PBS: phosphate buffered saline; PCK1: phosphoenolpyruvate carboxykinase 1, cytosolic; PCR: polymerase chain reaction; PFA: paraformaldehyde; RANBP9: RAN binding protein 9; RING: really interesting new gene; RMND5: required for meiotic nuclear division5 homolog; RPS6: ribosomal protein S6; RPTOR: regulatory associated protein of MTOR, complex 1; SE: short exposure; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TSC2: tuberous sclerosis complex 2; TUBA4A: tubulin; TUBE: tandem ubiquitin binding entities; Ub: ubiquitin; UPS: ubiquitin proteasome system; WDR26: WD repeat domain 26; WT: wild type.
Topics: AMP-Activated Protein Kinases; Adaptation, Physiological; Adenosine Triphosphate; Animals; Autophagy; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cilia; Longevity; Lysine; Mice; Multienzyme Complexes; NIH 3T3 Cells; Proteasome Endopeptidase Complex; Proteolysis; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitination
PubMed: 31795790
DOI: 10.1080/15548627.2019.1695399 -
Biomolecules Jun 2021The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of...
The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD-RIAD) system. These peptides allow self-assembly based on specific protein-protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer-Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMO-PTDH and PAMO-PTDH). These enzyme complexes displayed similar catalytic activity when compared with the PTDH-PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO-PTDH complex less PTDH was required for the same performance when compared with the PTDH-PAMO fusion enzyme.
Topics: Mixed Function Oxygenases; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Recombinant Proteins
PubMed: 34204515
DOI: 10.3390/biom11060905 -
Journal of the American Chemical Society Mar 2024Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical...
Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet changing cellular needs. However, replicating such capability in synthetic metabolons remains a challenge due to our limited understanding of the mechanisms by which the assembly and disassembly of such naturally occurring multienzyme complexes are controlled. Here, we report the synthesis of chemical- and light-responsive protein cages for assembling synthetic metabolons, enabling the dynamic regulation of enzymatic reactions in living cells. Particularly, a chemically responsive domain was fused to a self-assembled protein cage subunit, generating engineered protein cages capable of displaying proteins containing cognate interaction domains on their surfaces in response to small molecular cues. Chemical-induced colocalization of sequential enzymes on protein cages enhances the specificity of the branched deoxyviolacein biosynthetic reactions by 2.6-fold. Further, by replacing the chemical-inducible domain with a light-inducible dimerization domain, we created an optogenetic protein cage capable of reversibly recruiting and releasing targeted proteins onto and from the exterior of the protein cages in tens of seconds by on-off of blue light. Tethering the optogenetic protein cages to membranes enables the formation of light-switchable, membrane-bound metabolons, which can repeatably recruit-release enzymes, leading to the manipulation of substrate utilization across membranes on demand. Our work demonstrates a powerful and versatile strategy for constructing dynamic metabolons in engineered living cells for efficient and controllable biocatalysis.
Topics: Proteins; Multienzyme Complexes
PubMed: 38425051
DOI: 10.1021/jacs.3c12876 -
Experimental & Molecular Medicine Dec 2020Eukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting... (Review)
Review
Eukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).
Topics: Carrier Proteins; Cell Cycle Checkpoints; Chromatin; DNA Damage; DNA Repair; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Eukaryotic Cells; Genomic Instability; Multienzyme Complexes; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 33339954
DOI: 10.1038/s12276-020-00533-3 -
Extremophiles : Life Under Extreme... Jan 2022Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into...
Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A-G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes.
Topics: Aldehyde Oxidoreductases; Carbon Monoxide; Ecosystem; Multienzyme Complexes; Nickel; Phylogeny
PubMed: 35059858
DOI: 10.1007/s00792-022-01259-y -
Bioconjugate Chemistry Oct 2020Multienzyme complexes, or metabolons, are natural assemblies or clusters of sequential enzymes in biosynthesis. Spatial proximity of the enzyme active sites results in a...
Multienzyme complexes, or metabolons, are natural assemblies or clusters of sequential enzymes in biosynthesis. Spatial proximity of the enzyme active sites results in a substrate channeling effect, streamlines the cascade reaction, and increases the overall efficiency of the metabolic pathway. Engineers have constructed synthetic multienzyme complexes to acquire better control of the metabolic flux and a higher titer of the target product. As most of these complexes are assembled through orthogonal interactions or bioconjugation reactions, the number of enzymes to be assembled is limited by the number of orthogonal interaction or reaction pairs. Here, we utilized the (TMV) virus-like particle (VLP) as protein scaffold and orthogonal reactive protein pairs (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag) as linker modules to assemble three terpene biosynthetic enzymes in . The enzyme assembly switched on the production of amorpha-4,11-diene, whereas the product was undetectable in all the controls without assembly. This work demonstrates a facile strategy for constructing scaffolded catalytic nanomachineries to biosynthesize valuable metabolites in bacterial cells, and a unique assembly induced the switch-on mechanism in biosynthesis for the first time.
Topics: Biocatalysis; Biosynthetic Pathways; Escherichia coli; Genetic Engineering; Multienzyme Complexes; Terpenes; Tobacco Mosaic Virus; Virion
PubMed: 33001630
DOI: 10.1021/acs.bioconjchem.0c00476