A soluble form of ammonia monooxygenase in Nitrosomonas europaea

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Stefan Gilch ◽  
Ortwin Meyer ◽  
Ingo Schmidt
Keyword(s):  
Signal Sequence ◽  
Nitrosomonas Europaea ◽  
Soluble Form ◽  
Resonance Spectroscopy ◽  
Β Subunit ◽  
Ammonia Monooxygenase ◽  
Absorption Bands ◽  
Electron Para Magnetic Resonance ◽  
Α Subunit ◽  
Oxidation Of Ammonia

AbstractAmmonia monooxygenase (AMO) ofNitrosomonas europaeais a metalloenzyme that catalyzes the oxidation of ammonia to hydroxylamine. This study shows that AMO resides in the cytoplasm of the bacteria in addition to its location in the membrane and is distributed approximately equally in both subcellular fractions. AMO in both fractions catalyzes the oxidation of ammonia and binds [14C]acetylene, a mechanism-based inhibitor which specifically interacts with catalytically active AMO. Soluble AMO was purified 12-fold to electrophoretic homogeneity with a yield of 8%. AMO has a molecular mass of approximately 283 kDa with subunits of ca. 27 kDa (α-subunit, AmoA), ca. 42 kDa (β-subunit, AmoB), and ca. 24 kDa (γ-subunit, cytochromec1) in an α3β3γ3sub-unit structure. Different from the β-subunit of membrane-bound AMO, AmoB of soluble AMO possesses an N-terminal signal sequence. AMO contains Cu (9.4±0.6 mol per mol AMO), Fe (3.9±0.3 mol per mol AMO), and Zn (0.5 to 2.6 mol per mol AMO). Upon reduction the visible absorption spectrum of AMO reveals absorption bands characteristic of cytochromec. Electron para-magnetic resonance spectroscopy of air-oxidized AMO at 50 K shows a paramagnetic signal originating from Cu2+and at 10 K a paramagnetic signal characteristic of heme-Fe.

scholarly journals Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd

1999 ◽  
Vol 342 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Raymond M. D. VERHAERT ◽  
Jan VAN DUIN ◽  
Wim J. QUAX
Keyword(s):  
Coat Protein ◽  
Signal Sequence ◽  
Protein A ◽  
Alcaligenes Faecalis ◽  
Penicillin Acylase ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Β Subunit ◽  
Extracellular Medium ◽  
Α Subunit

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (α subunit-internal peptide-β subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the β-subunit is covalently linked and the α-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

scholarly journals Interaction of the mechanism-based inactivator acetylene with ammonia monooxygenase of Nitrosomonas europaea

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 279-284 ◽  
Author(s):  
Stefan Gilch ◽  
Manja Vogel ◽  
Matthias W. Lorenz ◽  
Ortwin Meyer ◽  
Ingo Schmidt
Keyword(s):  
Molecular Mass ◽  
Binding Site ◽  
Nitrosomonas Europaea ◽  
Ammonia Monooxygenase ◽  
Oxidation Of Ammonia

The ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyses the oxidation of ammonia to hydroxylamine. We have identified histidine 191 of AmoA as the binding site for the oxidized mechanism-based inactivator acetylene. Binding of acetylene changed the molecular mass of His-191 from 155.15 to 197.2 Da (+42.05), providing evidence that acetylene was oxidized to ketene (CH2CO; 42.04 Da) which binds specifically to His-191. It must be assumed that His-191 is part of the acetylene-activating site in AMO or at least directly neighbours this site.

Nevirapine Pharmaceutical Cocrystal: Design, Development and Formulation

2019 ◽  
Vol 9 (3) ◽  
pp. 240-247
Author(s):  
Prabhakar Panzade ◽  
Priyanka Somani ◽  
Pavan Rathi
Keyword(s):  
Dosage Form ◽  
In Vitro Dissolution ◽  
Soluble Form ◽  
Ir Absorption ◽  
Stoichiometric Ratio ◽  
Design Development ◽  
Formulation Development ◽  
Scanning Calorimetry ◽  
Absorption Bands ◽  
Pharmaceutical Cocrystal

Background and Objective: The top approach to deliver poorly soluble drugs is the use of a highly soluble form. The present study was conducted to enhance the solubility and dissolution of a poorly aqueous soluble drug nevirapine via a pharmaceutical cocrystal. Another objective of the study was to check the potential of the nevirapine cocrystal in the dosage form. Methods: A neat and liquid assisted grinding method was employed to prepare nevirapine cocrystals in a 1:1 and 1:2 stoichiometric ratio of drug:coformer by screening various coformers. The prepared cocrystals were preliminary investigated for melting point and saturation solubility. The selected cocrystal was further confirmed by Infrared Spectroscopy (IR), Differential Scanning Calorimetry (DSC), and Xray Powder Diffraction (XRPD). Further, the cocrystal was subjected to in vitro dissolution study and formulation development. Results: The cocrystal of Nevirapine (NVP) with Para-Amino Benzoic Acid (PABA) coformer prepared by neat grinding in 1:2 ratio exhibited greater solubility. The shifts in IR absorption bands, alterations in DSC thermogram, and distinct XRPD pattern showed the formation of the NVP-PABA cocrystal. Dissolution of NVP-PABA cocrystal enhanced by 38% in 0.1N HCl. Immediate release tablets of NVP-PABA cocrystal exhibited better drug release and less disintegration time. Conclusion: A remarkable increase in the solubility and dissolution of NVP was obtained through the cocrystal with PABA. The cocrystal also showed great potential in the dosage form which may provide future direction for other drugs.

scholarly journals Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA

2021 ◽  
Vol 22 (15) ◽  
pp. 7843
Author(s):  
Sang-Oh Ahn ◽  
Ho-Dong Lim ◽  
Sung-Hwan You ◽  
Dae-Eun Cheong ◽  
Geun-Joong Kim
Keyword(s):  
Tertiary Structure ◽  
Signal Sequence ◽  
Soluble Expression ◽  
Class I ◽  
Soluble Form ◽  
Two Phase ◽  
E Coli ◽  
Small Proteins ◽  
Industrial Use ◽  
Shed Light

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit, glycoprotein α-subunit, and thyroid hormone receptors α and β in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas

2009 ◽  
Vol 202 (1) ◽  
pp. 43-54 ◽  
Author(s):  
Sean C Lema ◽  
Jon T Dickey ◽  
Irvin R Schultz ◽  
Penny Swanson
Keyword(s):  
Thyroid Hormone ◽  
Pituitary Gland ◽  
Teleost Fish ◽  
Hormone Receptors ◽  
Pimephales Promelas ◽  
Early Gene ◽  
Thyroid Hormone Receptors ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3′-triiodo-l-thyronine (T3) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T3 elevated circulating total T3, while methimazole depressed plasma levels of total thyroxine (T4). In both sexes, this methimazole-induced reduction in T4 led to elevated mRNA abundance for thyrotropin β-subunit (tshβ) in the pituitary gland. Fish treated with T3 had elevated transcript levels for TR isoforms α and β (trα and trβ) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T3 elevated gene transcripts for tshβ, glycoprotein hormone α-subunit (gphα), and trβ, while not affecting trα levels. Taken together, these results demonstrate negative feedback of T4 on pituitary tshβ, identify trα and trβ as T3-autoinduced genes in the brain and liver, and provide new evidence that tshβ, gphα, and trβ are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

scholarly journals Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

2017 ◽  
Vol 474 (5) ◽  
pp. 751-769 ◽  
Author(s):  
M. Kristian Koski ◽  
Jothi Anantharajan ◽  
Petri Kursula ◽  
Prathusha Dhavala ◽  
Abhinandan V. Murthy ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Coiled Coil ◽  
Β Subunit ◽  
Dimerization Domain ◽  
Α Subunit ◽  
Protein Dimer ◽  
Symmetric Molecule ◽  
Asymmetric Shape ◽  
Proline Rich Peptide ◽  
Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

Differential Regulation of the Epithelial Sodium Channel (ENaC) in Rat Kidney, Lung and Distal Colon in Response to Aldosterone.

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 724-724
Author(s):  
Shyama M E Masilamani ◽  
Gheun-Ho Kim ◽  
Mark A Knepper
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Distal Colon ◽  
Β Subunit ◽  
Dietary Salt ◽  
Α Subunit ◽  
Salt Restriction ◽  
Γ Subunit ◽  
Dietary Salt Restriction ◽  
Epithelial Sodium Channel Enac

P170 The mineralocorticoid hormone, aldosterone increases renal tubule Na absorption via increases in the protein abundances of the α-subunit of the epithelial sodium channel (ENaC) and the 70 kDa form of the γ- subunit of ENaC (JCI 104:R19-R23). This study assesses the affect of dietary salt restriction on the regulation of the epithelial sodium channel (ENaC) in the lung and distal colon, in addition to kidney, using semiquantitative immunoblotting. Rats were placed initially on either a control Na intake (0.02 meq/day), or a low Na intake (0.2 meq/day) for 10 days. The low salt treated rats demonstrated an increase in plasma aldosterone levels at day 10 (control = 0.78 + 0.32 nM; Na restricted = 3.50 + 1.30 nM). In kidney homogenates, there were marked increases in the band density of the α-subunit of ENaC (286 % of control) and the 70 kDa form of γ-subunit of ENaC (262 % of control), but no increase in the abundance of the β-subunit of ENaC. In lung homogenates, there was no significant change in the band densities of the α, β, or γ subunits of ENaC. In distal colon, there was an increase in the band density of the β-subunit of ENaC (311 % of control) and an increase in both the 85 kDa (2355% of control) and 70 kDa (843 % of control) form of the γ subunit of ENaC in response to dietary Na restriction. However, there was no significant difference in the band density of the α-subunit of ENaC. These findings demonstrate tissue specific regulation of the three subunits of ENaC in response to dietary salt restriction.

scholarly journals The Extracellular Domain of the β2Integrin β Subunit (CD18) Is Sufficient forEscherichia coliHemolysin andAggregatibacter actinomycetemcomitansLeukotoxin Cytotoxic Activity

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Laura C. Ristow ◽  
Vy Tran ◽  
Kevin J. Schwartz ◽  
Lillie Pankratz ◽  
Andrew Mehle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Integrin Signaling ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Content Type ◽  
Animal Pathogens ◽  
Tract Infections

ABSTRACTTheEscherichia colihemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA fromAggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the β2integrin β subunit. Δβ2cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2subunit, but not to αL, αM, or αXin far-Western blots. Genetic complementation of Δβ2cells with either β2or β2with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2integrin signaling is not required for cytotoxicity. Finally, β2mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2cells are equally sensitive toStaphylococcus aureusα-toxin andProteus mirabilisHpmA. Our studies show two RTX toxins use the β2integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCEUrinary tract infections are one of the most common bacterial infections worldwide. UropathogenicEscherichia colistrains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenicEscherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the β2integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. TheE. colihemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.

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