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Non-canonical Amino Acid Substrates of E. coli Aminoacyl-tRNA Synthetases.
Hartman, MCT
Chembiochem : a European journal of chemical biology. 2022;(1):e202100299
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Abstract
In this comprehensive review, I focus on the twenty E. coli aminoacyl-tRNA synthetases and their ability to charge non-canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20 E. coli aminoacyl-tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.
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Escherichia coli small molecule metabolism at the host-microorganism interface.
Gatsios, A, Kim, CS, Crawford, JM
Nature chemical biology. 2021;(10):1016-1026
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Escherichia coli are a common component of the human microbiota, and isolates exhibit probiotic, commensal and pathogenic roles in the host. E. coli members often use diverse small molecule chemistry to regulate intrabacterial, intermicrobial and host-bacterial interactions. While E. coli are considered to be a well-studied model organism in biology, much of their chemical arsenal has only more recently been defined, and much remains to be explored. Here we describe chemical signaling systems in E. coli in the context of the broader field of metabolism at the host-bacteria interface and the role of this signaling in disease modulation.
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The Classical, Yet Controversial, First Enzyme of Lipid Synthesis: Escherichia coli Acetyl-CoA Carboxylase.
Cronan, JE
Microbiology and molecular biology reviews : MMBR. 2021;(3):e0003221
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Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA, the building block of fatty acid synthesis, is the paradigm bacterial ACC. Many reports on the structures and stoichiometry of the four subunits comprising the active enzyme as well as on regulation of ACC activity and expression have appeared in the almost 20 years since this subject was last reviewed. This review seeks to update and expand on these reports.
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Studying bacterial chemosensory array with CryoEM.
Qin, Z, Zhang, P
Biochemical Society transactions. 2021;(5):2081-2089
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Abstract
Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.
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5.
Copper tolerance in bacteria requires the activation of multiple accessory pathways.
Giachino, A, Waldron, KJ
Molecular microbiology. 2020;(3):377-390
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Copper is a required micronutrient for bacteria and an essential cofactor for redox-active cuproenzymes. Yet, excess copper is extremely toxic, and is exploited as a bacteriocide in medical and biotechnological applications and also by the mammalian immune system. To evade copper toxicity, bacteria not only control intracellular copper homeostasis, but they must also repair the damage caused by excess copper. In this review, we summarize the bacterial cell-wide response to copper toxicity in Enterobacteria. Tapping into the abundant research data on two key organisms, Escherichia coli and Salmonella enterica, we show that copper resistance requires both the direct copper homeostatic response and also the indirect accessory pathways that deal with copper-induced damage. Since patterns of copper response are conserved through the Proteobacteria, we propose a cell-wide view of copper detoxification and copper tolerance that can be used to identify novel targets for copper-based antibacterial therapeutics.
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Protein Disulfide Exchange by the Intramembrane Enzymes DsbB, DsbD, and CcdA.
Bushweller, JH
Journal of molecular biology. 2020;(18):5091-5103
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Abstract
The formation of disulfide bonds in proteins is an essential process in both prokaryotes and eukaryotes. In gram-negative bacteria including Escherichia coli, the proteins DsbA and DsbB mediate the formation of disulfide bonds in the periplasm. DsbA acts as the periplasmic oxidant of periplasmic substrate proteins. DsbA is reoxidized by transfer of reducing equivalents to the 4 TM helix membrane protein DsbB, which transfers reducing equivalents to ubiquinone or menaquinone. Multiple structural studies of DsbB have provided detailed structural information on intermediates in the process of DsbB catalyzed oxidation of DsbA. These structures and the insights gained are described. In proteins with more than one pair of Cys residues, there is the potential for formation of non-native disulfide bonds, making it necessary for the cell to have a mechanism for the isomerization of such non-native disulfide bonds. In E. coli, this is mediated by the proteins DsbC and DsbD. DsbC reduces mis-formed disulfide bonds. The eight-TM-helix protein DsbD reduces DsbC and is itself reduced by cytoplasmic thioredoxin. DsbD also contributes reducing equivalents for the reduction of cytochrome c to facilitate heme attachment. The DsbD functional homolog CcdA is a six-TM-helix membrane protein that provides reducing equivalents for the reduction of cytochrome c. A recent structure determination of CcdA has provided critical insights into how reducing equivalents are transferred across the membrane that likely also provides understanding how this is achieved by DsbD as well. This structure and the insights gained are described.
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The biosynthesis of the molybdenum cofactors in Escherichia coli.
Leimkühler, S
Environmental microbiology. 2020;(6):2007-2026
Abstract
The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5'-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.
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Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis.
Smolskaya, S, Logashina, YA, Andreev, YA
International journal of molecular sciences. 2020;(3)
Abstract
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
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Metabolic engineering of Escherichia coli for production of chemicals derived from the shikimate pathway.
Li, Z, Wang, H, Ding, D, Liu, Y, Fang, H, Chang, Z, Chen, T, Zhang, D
Journal of industrial microbiology & biotechnology. 2020;(6-7):525-535
Abstract
The shikimate pathway is indispensable for the biosynthesis of natural products with aromatic moieties. These products have wide current and potential applications in food, cosmetics and medicine, and consequently have great commercial value. However, compounds extracted from various plants or synthesized from petrochemicals no longer satisfy the requirements of contemporary industries. As a result, an increasing number of studies has focused on this pathway to enable the biotechnological manufacture of natural products, especially in E. coli. Furthermore, the development of synthetic biology, systems metabolic engineering and high flux screening techniques has also contributed to improving the biosynthesis of high-value compounds based on the shikimate pathway. Here, we review approaches based on a combination of traditional and new metabolic engineering strategies to increase the metabolic flux of the shikimate pathway. In addition, applications of this optimized pathway to produce aromatic amino acids and a range of natural products is also elaborated. Finally, this review sums up the opportunities and challenges facing this field.
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Enzymatic reactions and microorganisms producing the various isomers of hydroxyproline.
Hara, R, Kino, K
Applied microbiology and biotechnology. 2020;(11):4771-4779
Abstract
Hydroxyproline is an industrially important compound with applications in the pharmaceutical, nutrition, and cosmetic industries. trans-4-Hydroxy-L-proline is recognized as the most abundant of the eight possible isomers (hydroxy group at C-3 or C-4, cis- or trans-configuration, and L- or D-form). However, little attention has been paid to the rare isomers, probably due to their limited availability. This mini-review provides an overview of recent advances in microbial and enzymatic processes to develop practical production strategies for various hydroxyprolines. Here, we introduce three screening strategies, namely, activity-, sequence-, and metabolite-based approaches, allowing identification of diverse proline-hydroxylating enzymes with different product specificities. All naturally occurring hydroxyproline isomers can be produced by using suitable hydroxylases in a highly regio- and stereo-selective manner. Furthermore, crystal structures of relevant hydroxylases provide much insight into their functional roles. Since hydroxylases acting on free L-proline belong to the 2-oxoglutarate-dependent dioxygenase superfamily, cellular metabolism of Escherichia coli coupled with a hydroxylase is a valuable source of 2-oxoglutarate, which is indispensable as a co-substrate in L-proline hydroxylation. Further, microbial hydroxyproline 2-epimerase may serve as a highly adaptable tool to convert L-hydroxyproline into D-hydroxyproline. KEY POINTS • Proline hydroxylases serve as powerful tools for selectivel-proline hydroxylation. • Engineered Escherichia coli are a robust platform for hydroxyproline production. • Hydroxyproline epimerase convertsl-hydroxyproline intod-hydroxyproline.