scholarly journals A Single Tool to Monitor Multiple Protein–Protein Interactions of the Escherichia coli Acyl Carrier Protein

2019 ◽  
Vol 5 (9) ◽  
pp. 1518-1523 ◽  
Author(s):  
Katherine Charov ◽  
Michael D. Burkart
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Multiple Protein

Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain

2016 ◽  
Vol 128 (42) ◽  
pp. 13199-13203 ◽  
Author(s):  
Jesus F. Barajas ◽  
Kara Finzel ◽  
Timothy R. Valentic ◽  
Gaurav Shakya ◽  
Nathan Gamarra ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biochemical Analysis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Protein Protein Interactions

scholarly journals Activities and regulation of peptidoglycan synthases

2015 ◽  
Vol 370 (1679) ◽  
pp. 20150031 ◽  
Author(s):  
Alexander J. F. Egan ◽  
Jacob Biboy ◽  
Inge van't Veer ◽  
Eefjan Breukink ◽  
Waldemar Vollmer
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Cytoplasmic Membrane ◽  
Current Knowledge ◽  
Protein Protein Interactions ◽  
Penicillin Binding Proteins ◽  
Multiple Protein ◽  
Cell Division Protein ◽  
The Impact

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

scholarly journals Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

2020 ◽  
Author(s):  
Michael Burkart ◽  
Thomas Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D Lee ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Protein Docking ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Acid Biosynthesis ◽  
E Coli

Abstract Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This work reveals the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways. ONE SENTENCE SUMMARY: Through a combination of structural and computational analysis, a comparative evaluation of protein-protein interactions in de novo fatty acid biosynthesis in E. coli is performed.

scholarly journals Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6791
Author(s):  
Dongjin Leng ◽  
Yong Sheng ◽  
Hengyu Wang ◽  
Jianhua Wei ◽  
Yixin Ou ◽  
...  
Keyword(s):  
Mitomycin C ◽  
Protein Interactions ◽  
Biochemical Analysis ◽  
Genetic Manipulation ◽  
Acyl Carrier Protein ◽  
Early Stage ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Enzymatic Mechanisms

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein–protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein–protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein–protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

scholarly journals Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Thomas G. Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D. John Lee ◽  
Megan A. Young ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Protein Docking ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Docking Simulations ◽  
E Coli ◽  
Transient Nature ◽  
Partner Proteins

AbstractFatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

scholarly journals Decoding allosteric regulation by the acyl carrier protein

2021 ◽  
Vol 118 (16) ◽  
pp. e2025597118
Author(s):  
Terra Sztain ◽  
Thomas G. Bartholow ◽  
D. John Lee ◽  
Lorenzo Casalino ◽  
Andrew Mitchell ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Metabolic Pathways ◽  
Acyl Carrier Protein ◽  
Allosteric Regulation ◽  
Structural Features ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Fatty Acid Chain ◽  
Four Helical Bundle ◽  
Chain Lengths

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

scholarly journals Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function

1998 ◽  
Vol 62 (2) ◽  
pp. 309-333 ◽  
Author(s):  
Peter Stanley ◽  
Vassilis Koronakis ◽  
Colin Hughes
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Calcium Binding ◽  
Lipid A ◽  
Acyl Carrier Protein ◽  
Signal Transduction Pathway ◽  
Protein Protein Interactions ◽  
Lysine Residues ◽  
Acyl Chains ◽  
Escherichia Coli Hemolysin

SUMMARY The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a “double-anchor” motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation.

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