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Molecular Cell Feb 2023Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here,... (Review)
Review
Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.
Topics: Enhancer Elements, Genetic; Transcription Factors; Promoter Regions, Genetic; Gene Expression Regulation; Base Sequence; Chromatin
PubMed: 36693380
DOI: 10.1016/j.molcel.2022.12.032 -
Chemical Society Reviews Jul 2018The continuous flow synthesis of active pharmaceutical ingredients, value-added chemicals, and materials has grown tremendously over the past ten years. This revolution... (Review)
Review
The continuous flow synthesis of active pharmaceutical ingredients, value-added chemicals, and materials has grown tremendously over the past ten years. This revolution in chemical manufacturing has resulted from innovations in both new methodology and technology. This field, however, has been predominantly focused on synthetic organic chemistry, and the use of biocatalysts in continuous flow systems is only now becoming popular. Although immobilized enzymes and whole cells in batch systems are common, their continuous flow counterparts have grown rapidly over the past two years. With continuous flow systems offering improved mixing, mass transfer, thermal control, pressurized processing, decreased variation, automation, process analytical technology, and in-line purification, the combination of biocatalysis and flow chemistry opens powerful new process windows. This Review explores continuous flow biocatalysts with emphasis on new technology, enzymes, whole cells, co-factor recycling, and immobilization methods for the synthesis of pharmaceuticals, value-added chemicals, and materials.
Topics: Biocatalysis; Bioreactors; Cells, Immobilized; Enzyme Activation; Enzymes, Immobilized; Humans; Pharmaceutical Preparations; Physical Phenomena; Pressure; Surface Properties; Technology, Pharmaceutical; Temperature
PubMed: 29922795
DOI: 10.1039/c7cs00906b -
Microbial Cell Factories Jun 2017Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as... (Review)
Review
Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals.
Topics: Bacteria; Biocatalysis; Biosynthetic Pathways; Coenzymes; Metabolic Engineering; Organic Chemicals; Synthetic Biology
PubMed: 28610636
DOI: 10.1186/s12934-017-0724-7 -
Angewandte Chemie (International Ed. in... Jul 2022Cofactors are required for almost half of all enzyme reactions, but their functions and binding partners are not fully understood even after decades of research.... (Review)
Review
Cofactors are required for almost half of all enzyme reactions, but their functions and binding partners are not fully understood even after decades of research. Functionalised cofactor mimics that bind in place of the unmodified cofactor can provide answers, as well as expand the scope of cofactor activity. Through chemical proteomics approaches such as activity-based protein profiling, the interactome and localisation of the native cofactor in its physiological environment can be deciphered and previously uncharacterised proteins annotated. Furthermore, cofactors that supply functional groups to substrate biomolecules can be hijacked by mimics to site-specifically label targets and unravel the complex biology of post-translational protein modification. The diverse activity of cofactors has inspired the design of mimics for use as inhibitors, antibiotic therapeutics, and chemo- and biosensors, and cofactor conjugates have enabled the generation of novel enzymes and artificial DNAzymes.
Topics: DNA, Catalytic; Proteomics
PubMed: 35286003
DOI: 10.1002/anie.202201136 -
Nucleic Acids Research Jun 2023Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of...
Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6-GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.
Topics: Animals; Humans; Mice; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Erythroid Cells; Erythropoiesis; GATA1 Transcription Factor; Gene Expression Profiling; Polycythemia Vera; Repressor Proteins
PubMed: 36929421
DOI: 10.1093/nar/gkad167 -
International Journal of Molecular... May 2021The production of aldehydes, highly reactive and toxic chemicals, brings specific challenges to biocatalytic processes. Absence of natural accumulation of aldehydes in... (Review)
Review
The production of aldehydes, highly reactive and toxic chemicals, brings specific challenges to biocatalytic processes. Absence of natural accumulation of aldehydes in microorganisms has led to a combination of in vitro and in vivo strategies for both, bulk and fine production. Advances in genetic and metabolic engineering and implementation of computational techniques led to the production of various enzymes with special requirements. Cofactor synthesis, post-translational modifications and structure engineering are applied to prepare active enzymes for one-step or cascade reactions. This review presents the highlights in biocatalytical production of aldehydes with the potential to shape future industrial applications.
Topics: Aldehydes; Biocatalysis; Biosensing Techniques; Enzymes; Ligands; Metabolic Engineering
PubMed: 34066641
DOI: 10.3390/ijms22094949 -
ACS Bio & Med Chem Au Aug 2022Radical -adenosylmethionine (RaS) enzymes have quickly advanced to one of the most abundant and versatile enzyme superfamilies known. Their chemistry is predicated upon... (Review)
Review
Radical -adenosylmethionine (RaS) enzymes have quickly advanced to one of the most abundant and versatile enzyme superfamilies known. Their chemistry is predicated upon reductive homolytic cleavage of a carbon-sulfur bond in cofactor -adenosylmethionine forming an oxidizing carbon-based radical, which can initiate myriad radical transformations. An emerging role for RaS enzymes is their involvement in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a natural product family that has become known as RaS-RiPPs. These metabolites are especially prevalent in human and mammalian microbiomes because the complex chemistry of RaS enzymes gives rise to correspondingly complex natural products with minimal cellular energy and genomic fingerprint, a feature that is advantageous in microbes with small, host-adapted genomes in competitive environments. Herein, we review the discovery and characterization of RaS-RiPPs from the human microbiome with a focus on streptococcal bacteria. We discuss the varied chemical modifications that RaS enzymes introduce onto their peptide substrates and the diverse natural products that they give rise to. The majority of RaS-RiPPs remain to be discovered, providing an intriguing avenue for future investigations at the intersection of metalloenzymology, chemical ecology, and the human microbiome.
PubMed: 35996476
DOI: 10.1021/acsbiomedchemau.2c00004 -
Nature Communications Feb 2024Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F. Despite decades of research, key...
Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.
Topics: Humans; Glutamic Acid; Folic Acid; Peptide Synthases; Bacteria; Protein Processing, Post-Translational
PubMed: 38346985
DOI: 10.1038/s41467-024-45632-1 -
ELife Sep 2023The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and...
The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members CP nuclear ofactor amily member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
Topics: Humans; Valosin Containing Protein; Nuclear Proteins; Active Transport, Cell Nucleus
PubMed: 37713320
DOI: 10.7554/eLife.92409 -
ChemSusChem Nov 2022Enzymatic processes, particularly those capable of performing redox reactions, have recently been of growing research interest. Substrate specificity, optimal activity... (Review)
Review
Enzymatic processes, particularly those capable of performing redox reactions, have recently been of growing research interest. Substrate specificity, optimal activity at mild temperatures, high selectivity, and yield are among the desirable characteristics of these oxidoreductase catalyzed reactions. Nicotinamide adenine dinucleotide (phosphate) or NAD(P)H-dependent oxidoreductases have been extensively studied for their potential applications like biosynthesis of chiral organic compounds, construction of biosensors, and pollutant degradation. One of the main challenges associated with making these processes commercially viable is the regeneration of the expensive cofactors required by the enzymes. Numerous efforts have pursued enzymatic regeneration of NAD(P)H by coupling a substrate reduction with a complementary enzyme catalyzed oxidation of a co-substrate. While offering excellent selectivity and high total turnover numbers, such processes involve complicated downstream product separation of a primary product from the coproducts and impurities. Alternative methods comprising chemical, electrochemical, and photochemical regeneration have been developed with the goal of enhanced efficiency and operational simplicity compared to enzymatic regeneration. Despite the goal, however, the literature rarely offers a meaningful comparison of the total turnover numbers for various regeneration methodologies. This comprehensive Review systematically discusses various methods of NAD(P)H cofactor regeneration and quantitatively compares performance across the numerous methods. Further, fundamental barriers to enhanced cofactor regeneration in the various methods are identified, and future opportunities are highlighted for improving the efficiency and sustainability of commercially viable oxidoreductase processes for practical implementation.
Topics: Biocatalysis; NAD; Niacinamide; Oxidation-Reduction; NADP; Oxidoreductases; Regeneration
PubMed: 36129761
DOI: 10.1002/cssc.202200888