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ACS Synthetic Biology Oct 2022Nature is enriched with specific interactions between receptor proteins and their cognate ligands. These interacting pairs can be exploited and applied for the... (Review)
Review
Nature is enriched with specific interactions between receptor proteins and their cognate ligands. These interacting pairs can be exploited and applied for the construction of well-ordered multicomponent assemblies with multivalency and multifunctionality. One of the research hotspots of this area is the formation of multienzyme complexes with stable and tunable architectures, which may bear the potential to facilitate cascade biocatalysis and/or strengthen metabolic fluxes. Here we focus on a special interacting pair, the anchoring domain (AD) derived from A-kinase anchoring protein and its interacting dimerization and docking domain (DDD) derived from cyclic AMP-dependent protein kinase, which has potential to be an effective and powerful synthetic biology tool for the construction of multienzyme assemblies. We review the origin and interaction mechanism of AD-DDD, followed by the application of this so-called dock-and-lock pair to form various bioconjugates with multivalency and multispecificity. Then several recent studies related to the construction of multienzyme complexes using AD-DDD, and more specifically, the RIAD-RIDD interacting pair, are presented. Finally, we also discuss the great biotechnology potential and perspectives of AD-DDD as a potent synthetic biology tool for post-translational modifications.
Topics: A Kinase Anchor Proteins; Cyclic AMP-Dependent Protein Kinases; Dimerization; Synthetic Biology; Ligands; Multienzyme Complexes
PubMed: 36197832
DOI: 10.1021/acssynbio.2c00443 -
Molecules (Basel, Switzerland) Mar 2021Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these... (Review)
Review
Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial production of chemicals, biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biology methodologies, to complement the purposeful deployment of enzymes. Current molecular tools for constructing these synthetic enzyme-scaffold systems are also highlighted.
Topics: Animals; Biocatalysis; Enzyme Stability; Enzymes; Genetic Therapy; Humans; Multienzyme Complexes; Protein Engineering; Synthetic Biology
PubMed: 33806660
DOI: 10.3390/molecules26051389 -
The FEBS Journal Aug 2022Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin,... (Review)
Review
Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin, and insulin, each of which is stored in electron-dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the ER lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by peptidylglycine α-amidating monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes, and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell types.
Topics: Chlamydomonas reinhardtii; Humans; Mixed Function Oxygenases; Multienzyme Complexes; Myocytes, Cardiac; Neuroendocrine Cells; Peptides; Phylogeny
PubMed: 34089560
DOI: 10.1111/febs.16049 -
Annual Review of Virology Sep 2019RNA turnover and processing in bacteria are governed by the structurally divergent but functionally convergent RNA degradosome, and the mechanisms have been researched... (Review)
Review
RNA turnover and processing in bacteria are governed by the structurally divergent but functionally convergent RNA degradosome, and the mechanisms have been researched extensively in Gram-positive and Gram-negative bacteria. An emerging research field focuses on how bacterial viruses hijack all aspects of the bacterial metabolism, including the host machinery of RNA metabolism. This review addresses research on phage-based influence on RNA turnover, which can act either indirectly or via dedicated effector molecules that target degradosome assemblies. The structural divergence of host RNA turnover mechanisms likely explains the limited number of phage proteins directly targeting these specialized, host-specific complexes. The unique and nonconserved structure of DIP, a phage-encoded inhibitor of the degradosome, illustrates this hypothesis. However, the natural occurrence of phage-encoded mechanisms regulating RNA turnover indicates a clear evolutionary benefit for this mode of host manipulation. Further exploration of the viral dark matter of unknown phage proteins may reveal more structurally novel interference strategies that, in turn, could be exploited for biotechnological applications.
Topics: Bacteriophages; Endoribonucleases; Gene Expression Regulation, Viral; Gram-Negative Bacteria; Gram-Positive Bacteria; Host Microbial Interactions; Multienzyme Complexes; Polyribonucleotide Nucleotidyltransferase; RNA Helicases; RNA, Bacterial
PubMed: 31100993
DOI: 10.1146/annurev-virology-092818-015644 -
EcoSal Plus Jan 2020Two-component regulatory systems represent the major paradigm for signal transduction in prokaryotes. The simplest systems are composed of a sensor kinase and a response... (Review)
Review
Two-component regulatory systems represent the major paradigm for signal transduction in prokaryotes. The simplest systems are composed of a sensor kinase and a response regulator. The sensor is often a membrane protein that senses a change in environmental conditions and is autophosphorylated by ATP on a histidine residue. The phosphoryl group is transferred onto an aspartate of the response regulator, which activates the regulator and alters its output, usually resulting in a change in gene expression. In this review, we present a historical view of the archetype EnvZ/OmpR two-component signaling system, and then we provide a new view of signaling based on our recent experiments. EnvZ responds to cytoplasmic signals that arise from changes in the extracellular milieu, and OmpR acts canonically (requiring phosphorylation) to regulate the porin genes and noncanonically (without phosphorylation) to activate the acid stress response. Herein, we describe how insights gleaned from stimulus recognition and response in EnvZ are relevant to nearly all sensor kinases and response regulators.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Multienzyme Complexes; Phosphorylation; Signal Transduction; Trans-Activators
PubMed: 32003321
DOI: 10.1128/ecosalplus.ESP-0001-2019 -
International Journal of Biological... Jul 2022Energy metabolism is a universal process occurring in all life forms. In Mycobacterium tuberculosis (Mtb), energy production is carried out in two possible ways,... (Review)
Review
Energy metabolism is a universal process occurring in all life forms. In Mycobacterium tuberculosis (Mtb), energy production is carried out in two possible ways, oxidative phosphorylation (OxPhos) and substrate-level phosphorylation. Mtb is an obligate aerobic bacterium, making it dependent on OxPhos for ATP synthesis and growth. Mtb inhabits varied micro-niches during the infection cycle, outside and within the host cells, which alters its primary metabolic pathways during the pathogenesis. In this review, we discuss cellular respiration in the context of the mechanism and structural importance of the proteins and enzyme complexes involved. These protein-protein complexes have been proven to be essential for Mtb virulence as they aid the bacteria's survival during aerobic and hypoxic conditions. ATP synthase, a crucial component of the electron transport chain, has been in the limelight, as a prominent drug target against tuberculosis. Likewise, in this review, we have explored other protein-protein complexes of the OxPhos pathway, their functional essentiality, and their mechanism in Mtb's diverse lifecycle. The review summarises crucial target proteins and reported inhibitors of the electron transport chain pathway of Mtb.
Topics: Adenosine Triphosphate; Electron Transport; Humans; Multienzyme Complexes; Mycobacterium tuberculosis; Tuberculosis
PubMed: 35613677
DOI: 10.1016/j.ijbiomac.2022.05.124 -
ACS Nano Sep 2019Multienzyme complexes, or metabolons, are assemblies or clusters of sequential enzymes that naturally exist in metabolic pathways. These nanomachineries catalyze the...
Multienzyme complexes, or metabolons, are assemblies or clusters of sequential enzymes that naturally exist in metabolic pathways. These nanomachineries catalyze the conversion of metabolites more effectively than the freely floating enzymes by minimizing the diffusion of intermediates . Bioengineers have devised synthetic versions of multienzyme complexes in cells to synergize heterologous biosynthesis, to improve intracellular metabolic flux, and to achieve higher titer of valuable chemical products. Here, we utilized orthogonal protein reactions (SpyCatcher/SpyTag and SnoopCatcher/SnoopTag pairs) to covalently assemble three key enzymes in the mevalonate biosynthesis pathway and showed 5-fold increase of lycopene and 2-fold increase of astaxanthin production in . The multienzyme complexes are ellipsoidal nanostructures with hollow interior space and uniform thickness and shapes. Intracellular covalent enzyme assembly has yielded catalytic nanomachineries that drastically enlarged the flux of carotenoid biosynthesis . These studies also deepened our understanding on the complexity of hierarchical enzyme assembly .
Topics: Amino Acid Sequence; Biocatalysis; Biosynthetic Pathways; Carotenoids; Multienzyme Complexes; Nanostructures; Proteins
PubMed: 31356751
DOI: 10.1021/acsnano.9b03631 -
Nature Mar 2024In eukaryotes, DNA compacts into chromatin through nucleosomes. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the...
In eukaryotes, DNA compacts into chromatin through nucleosomes. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.
Topics: Binding Sites; Chromatin; Cryoelectron Microscopy; DNA Replication; DNA, Fungal; Epistasis, Genetic; Histones; Multienzyme Complexes; Nucleosomes; Protein Binding; Protein Domains; Protein Multimerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38448592
DOI: 10.1038/s41586-024-07152-2 -
ACS Nano Oct 2019Sequential enzymes in a biosynthetic pathway often self-assemble to form nanomachineries known as multienzyme complexes inside cells. Enzyme self-assembly insulates...
Sequential enzymes in a biosynthetic pathway often self-assemble to form nanomachineries known as multienzyme complexes inside cells. Enzyme self-assembly insulates toxic intermediates, increases the efficiency of intermediate transfer, minimizes metabolic crosstalk, streamlines flux, and improves the product yield. Artful structures and superior catalytic functions of these natural nanomachines inspired the development of synthetic multienzyme complexes to expedite biosynthesis. Here we present a versatile self-assembly strategy to construct multienzyme nanostructures based on synthetic protein scaffolds. The protein scaffolds were formed using the spontaneous protein reaction of SpyCatcher and SpyTag. Two types of protein scaffolds were generated: two skeleton proteins cross-linked and hierarchically assembled into heterogeneous nanostructures (the cross-linked scaffold), and head-to-tail cyclization of a dual-reactive skeleton protein gave a homogeneous cyclic scaffold. Sequential enzymes from the menaquinone biosynthetic pathway were assembled on both scaffolds through the docking domain interactions derived from polyketide synthases. Both scaffolded assemblies effectively increased the yield of the final product of the cascade catalytic reaction in menaquinone biosynthesis. Surprisingly, the rate enhancements were driven by different mechanisms: the cross-linked scaffold assembly streamlined the overall flow of the reactants, whereas the cyclic scaffold assembly accelerated the catalytic efficiency of the rate-limiting enzyme. Altogether, self-assembly of sequential enzymes by combining the SpyCatcher/SpyTag reaction and the docking domain interactions yielded protein-based nanostructures with special architecture, exceptional catalytic activity, and unexpected catalytic mechanisms. This work demonstrates a versatile strategy of gaining more powerful biocatalysts by protein self-assembly for efficient bioconversion of valuable chemicals.
Topics: Models, Molecular; Multienzyme Complexes; Nanostructures; Proteins
PubMed: 31498583
DOI: 10.1021/acsnano.9b04554 -
Toxicology Sep 2019Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is... (Review)
Review
Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is critical for maintenance of pregnancy. HSD3B1 is located in the mitochondrion and the smooth endoplasmic reticulum of placental cells and is encoded by HSD3B1 gene. HSD3B1 contains GATA and TEF-5 regulatory elements. Many endocrine disruptors, including phthalates, methoxychlor and its metabolite, organotins, and gossypol directly inhibit placental HSD3B1 thus blocking progesterone production. In this review, we discuss the placental HSD3B1, its gene regulation, biochemistry, subcellular location, and inhibitors from the environment.
Topics: Environmental Pollutants; Female; Gene Expression Regulation; Humans; Multienzyme Complexes; Placenta; Pregnancy; Progesterone Reductase; Steroid Isomerases
PubMed: 31351905
DOI: 10.1016/j.tox.2019.152253