MpaA is a murein-tripeptide-specific zinc carboxypeptidase that functions as part of a catabolic pathway for peptidoglycan-derived peptides in γ-proteobacteria

2012 ◽  
Vol 448 (3) ◽  
pp. 329-341 ◽  
Author(s):  
Abbas Maqbool ◽  
Mireille Hervé ◽  
Dominique Mengin-Lecreulx ◽  
Anthony J. Wilkinson ◽  
Gavin H. Thomas
Keyword(s):  
Structural Similarity ◽  
Zinc Ion ◽  
Diaminopimelic Acid ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Catabolic Pathway ◽  
E Coli ◽  
Transporter Protein ◽  
Exclusion Chromatography ◽  
Binding Groove

The murein peptide amidase MpaA is a cytoplasmic enzyme that processes peptides derived from the turnover of murein. We have purified the enzyme from Escherichia coli and demonstrated that it efficiently hydrolyses the γ-D-glutamyl-diaminopimelic acid bond in the murein tripeptide (L-Ala-γ-D-Glu-meso-Dap), with Km and kcat values of 0.41±0.05 mM and 38.3±10 s−1. However, it is unable to act on the murein tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-D-Ala). E. coli MpaA is a homodimer containing one bound zinc ion per chain, as judged by mass spectrometric analysis and size-exclusion chromatography. To investigate the structure of MpaA we solved the crystal structure of the orthologous protein from Vibrio harveyi to 2.17 Å (1Å=0.1 nm). Vh_MpaA, which has identical enzymatic and biophysical properties to the E. coli enzyme, has high structural similarity to eukaryotic zinc carboxypeptidases. The structure confirms that MpaA is a dimeric zinc metalloprotein. Comparison of the structure of MpaA with those of other carboxypeptidases reveals additional structure that partially occludes the substrate-binding groove, perhaps explaining the narrower substrate specificity of the enzyme compared with other zinc carboxypeptidases. In γ-proteobacteria mpaA is often located adjacent to mppA which encodes a periplasmic transporter protein previously shown to bind murein tripeptide. We demonstrate that MppA can also bind murein tetrapeptide with high affinity. The genetic coupling of these genes and their related biochemical functions suggest that MpaA amidase and MppA transporter form part of a catabolic pathway for utilization of murein-derived peptides that operates in γ-proteobacteria in addition to the established murein recycling pathways.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

2017 ◽  
Vol 8 (5) ◽  
pp. 4062-4072 ◽  
Author(s):  
Michael Cammarata ◽  
Ross Thyer ◽  
Michael Lombardo ◽  
Amy Anderson ◽  
Dennis Wright ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Dihydrofolate Reductase ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
E Coli ◽  
Exclusion Chromatography

Native mass spectrometry, size exclusion chromatography, and kinetic assays were employed to study trimethoprim resistance in E. coli caused by mutations P21L and W30R of dihydrofolate reductase.

scholarly journals Characterization of Sorbitol Dehydrogenase SmoS fromSinorhizobium meliloti1021

2019 ◽  
Author(s):  
MacLean G. Kohlmeier ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Ivan J. Oresnik
Keyword(s):  
Sinorhizobium Meliloti ◽  
Model Organism ◽  
Time Frame ◽  
Industrial Applications ◽  
Size Exclusion ◽  
Ligand Docking ◽  
X Ray Diffraction ◽  
E Coli ◽  
Exclusion Chromatography

AbstractSinorhizobium meliloti1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+as a cofactor. The main objective of this study is to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon optimized for heterologous expression inE. coliBL21 (DE3) GOLD cells, the protein was subsequently overexpressed and purified. Size exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetrameric peptide. SmoS was crystallized to 2.1 Å in the absence of substrate and 2.0 Å in complex with sorbitol. SmoS was characterized kinetically and shown to have a preference for sorbitol despite a higher affinity for galactitol. Computational ligand docking experiments suggest that galactitol oxidation proceeds slowly because tagatose binds the protein in a more energetically favorable complex than fructose, and is retained in the active site for a longer time frame following oxidation which reduces the rate of the reaction. These results supplement the inventory of biomolecules with the potential for industrial applications and enhance our understanding of metabolism in the model organismS. meliloti.

Abstract 255: Plasminogen Promotes Cholesterol Efflux by the ABCA1

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Nathalie Pamir ◽  
David A Dichek ◽  
Godfrey S Getz ◽  
Santica Marcovina ◽  
Jay W Heinecke
Keyword(s):  
Size Exclusion Chromatography ◽  
Cholesterol Efflux ◽  
Particle Concentration ◽  
Specific Activity ◽  
Cholesterol Homeostasis ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Cvd Risk ◽  
Exclusion Chromatography

Background: The cholesterol efflux capacity (CEC) of serum HDL, measured using cultured macrophages predicts incident and prevalent CVD risk in humans. The ABCA1 pathway is a key regulator of macrophage cholesterol homeostasis in vivo. Methods: We used genetic and biochemical approaches in mice to identify important mediators of CEC. Results: On high-resolution size-exclusion chromatography of mouse plasma, macrophage CEC and HDL co-eluted as a single major peak, suggesting that HDL mediates cholesterol efflux. In contrast, size-exclusion chromatography revealed two major peaks of material that promoted ABCA1-specific CEC, one of which was distinct from HDL. HDL particle concentration was reduced by 75% in Apoa1 -/- mice; this resulted in a 50% decrease in macrophage CEC but, surprisingly, had no impact on ABCA1-specific CEC. Orthogonal chromatography-mass spectrometric analysis of the non-HDL-associated efflux inducing material isolated from wild-type and APOA1 deficient plasma showed that plasminogen strongly correlated with ABCA1-specific CEC. Moreover, isolated plasminogen promoted cholesterol efflux by the ABCA1 pathway, and the specific activity of ABCA1-specific CEC of non-HDL-associated material was reduced by 50% in plasminogen deficient plasma. Imaging of cells treated with fluorescently-labeled antibodies demonstrated that ABCA1 and plasminogen co-localized on the plasma membrane. Conclusions: HDL particle concentration is an important contributor to macrophage CEC. However, other pathways contribute to ABCA1-specific CEC; our studies identify plasminogen as one potential mediator. Plasminogen associates with CVD risk in human genetic studies, raising the possibility that it plays a role in atherosclerosis by modulating ABCA1-mediated sterol efflux from macrophages.

scholarly journals Crystal structure of an anti-idiotype variable lymphocyte receptor

2017 ◽  
Vol 73 (12) ◽  
pp. 682-687
Author(s):  
Bernard C. Collins ◽  
Hiro Nakahara ◽  
Sharmistha Acharya ◽  
Max D. Cooper ◽  
Brantley R. Herrin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Similarity ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Size Exclusion ◽  
Antigen Receptors ◽  
Diagnostic Assays ◽  
Great Utility ◽  
Exclusion Chromatography ◽  
Leukemic Clone

Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39–BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.

scholarly journals Serendipitous crystallization of E. coli HPII catalase, a sequel to “the tale usually not told”

2021 ◽  
Author(s):  
Marta Grzechowiak ◽  
Bartosz Sekula ◽  
Mariusz Jaskolski ◽  
Milosz Ruszkowski
Keyword(s):  
Molecular Weight ◽  
Carbonic Anhydrase ◽  
Recombinant Protein ◽  
Metal Binding ◽  
Recombinant Protein Expression ◽  
Crystal Form ◽  
Size Exclusion ◽  
E Coli ◽  
Low Concentrations ◽  
Exclusion Chromatography

Protein crystallographers are well aware of the trap of crystallizing E. coli proteins instead of the macromolecule of interest if heterologous recombinant protein expression in E. coli was part of the experimental pipeline. Among the well-known culprits are YodA metal-binding lipocalin (25 kDa) and YadF carbonic anhydrase (a tetramer of 25 kDa subunits). We report a novel crystal form of another such culprit, E. coli HPII catalase, which is a tetrameric protein of ~340 kDa molecular weight. HPII is likely to contaminate recombinant protein samples, co-purify, and then co-crystallize with the target proteins, especially if their masses in size exclusion chromatography are ~300–400 kDa. What makes this case more interesting but also parlous, is the fact that HPII can crystallize from very low concentrations, even well below 1 mg/mL.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis ofN-acetylmannosamine kinase from methicillin-resistantStaphylococcus aureus

2014 ◽  
Vol 70 (5) ◽  
pp. 643-649 ◽  
Author(s):  
Rachel A. North ◽  
Simona Seizova ◽  
Anja Stampfli ◽  
Sarah A. Kessans ◽  
Hironori Suzuki ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Bacterial Pathogens ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Sialic Acids ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
E Coli ◽  
Exclusion Chromatography

N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such asEscherichia coli,Staphylococcus aureus,Vibrio choleraeandV. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the `Nan-Nag cluster', which have proven to be essential forS. aureusgrowth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzymeN-acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine-5′-triphosphate to the C6 position ofN-acetylmannosamine to generateN-acetylmannosamine-6-phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed inE. coliBL21 (DE3) cells and purifiedviaanion-exchange chromatography, hydrophobic interaction chromatography and size-exclusion chromatography. PurifiedN-acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space groupP2. Understanding the structural nature of this enzyme from methicillin-resistantS. aureuswill provide insights necessary for the development of future antimicrobials.

scholarly journals Complex-centric proteome profiling by SEC-SWATH-MS for the parallel detection of hundreds of protein complexes

2019 ◽  
Author(s):  
Isabell Bludau ◽  
Moritz Heusel ◽  
Max Frank ◽  
George Rosenberger ◽  
Robin Hafen ◽  
...  
Keyword(s):  
Error Control ◽  
Complex Analysis ◽  
De Novo ◽  
Protein Complexes ◽  
Statistical Error ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Native Protein ◽  
Proteome Profiling ◽  
Exclusion Chromatography

Abstract Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules is therefore of major significance in basic and translational biology. To date, the systematic detection and quantification of protein complexes has remained technically challenging. The chromatographic separation of native protein complexes followed by the mass spectrometric analysis of the proteins contained in sequential fractions results in potentially thousands of protein elution profiles from which, in principle, the presence of specific complexes can be inferred. However, the de novo inference of protein complexes from such datasets has so far remained limited with regard to selectivity and the retrieval of quantitative information.We recently developed a variant of this strategy, complex-centric proteome profiling, which extends the concepts of targeted proteomics to the level of native protein complex analysis. The complex-centric workflow consists of size exclusion chromatography (SEC) to fractionate native protein complexes, DIA/SWATH mass spectrometry to precisely quantify the proteins in each SEC fraction based on a consistent set of peptides, and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler. Complex-centric proteome profiling captures the majority of proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to genetically unaltered tissue cultures and adaptable to primary tissue. At present it requires approximately 8 days of wet-lab work, 15 days of MS measurement time and 7 days of computational analysis.

scholarly journals gly Gene Cloning and Expression and Purification of Glycinecin A, a Bacteriocin Produced by Xanthomonas campestris pv. glycines 8ra

2001 ◽  
Vol 67 (9) ◽  
pp. 4105-4110 ◽  
Author(s):  
Sunggi Heu ◽  
Jonghee Oh ◽  
Youngsung Kang ◽  
Sangryeol Ryu ◽  
Somi K. Cho ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Culture Media ◽  
Cloning And Expression ◽  
Size Exclusion ◽  
Gene Cloning And Expression ◽  
E Coli ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Exclusion Chromatography ◽  
Wide Ph Range

ABSTRACT Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes,glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences ofglyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with eitherglyA or glyB separately did not show the bacteriocin activity.

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