Crystal structures of the amino-terminal domain of LpoA from Escherichia coli and Haemophilus influenzae

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Å and was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Å resolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.

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Crystal Structures of New Ivermectin Pseudopolymorphs

Crystals ◽

10.3390/cryst11020172 ◽

2021 ◽

Vol 11 (2) ◽

pp. 172

Author(s):

Kirill Shubin ◽

Agris Bērziņš ◽

Sergey Belyakov

Keyword(s):

Crystal Structure ◽

Molecular Conformation ◽

Methyl Tert Butyl Ether ◽

Minor Role ◽

Scanning Calorimetry ◽

Orthorhombic Crystal ◽

Monoclinic Crystal ◽

Orthorhombic Crystal Structure ◽

Space Group ◽

Structure Space

New pseudopolymorphs of ivermectin (IVM), a potential anti-COVID-19 drug, were prepared. The crystal structure for three pseudopolymorphic crystalline forms of IVM has been determined using single-crystal X-ray crystallographic analysis. The molecular conformation of IVM in crystals has been compared with the conformation of isolated molecules modeled by DFT calculations. In a solvent with relatively small molecules (ethanol), IVM forms monoclinic crystal structure (space group I2), which contains two types of voids. When crystallized from solvents with larger molecules, like γ-valerolactone (GVL) and methyl tert-butyl ether (MTBE), IVM forms orthorhombic crystal structure (space group P212121). Calculations of the lattice energy indicate that interactions between IVM and solvents play a minor role; the main contribution to energy is made by the interactions between the molecules of IVM itself, which form a framework in the crystal structure. Interactions between IVM and molecules of solvents were evaluated using Hirshfeld surface analysis. Thermal analysis of the new pseudopolymorphs of IVM was performed by differential scanning calorimetry and thermogravimetric analysis.

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Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.187.20.6928-6935.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 6928-6935 ◽

Cited By ~ 9

Author(s):

Valley Stewart ◽

Peggy J. Bledsoe

Keyword(s):

Escherichia Coli ◽

Nitrate Reductase ◽

Haemophilus Influenzae ◽

Control Region ◽

Binding Sites ◽

Transcription Initiation ◽

Transcriptional Control ◽

Class I ◽

E Coli ◽

Fnr Protein

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

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Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases

Applied and Environmental Microbiology ◽

10.1128/aem.69.6.3448-3455.2003 ◽

2003 ◽

Vol 69 (6) ◽

pp. 3448-3455 ◽

Cited By ~ 84

Author(s):

JÃ¯Â¿Â½rg Rau ◽

Andreas Stolz

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Bacterial Species ◽

Azo Compounds ◽

Biochemical Tests ◽

Pronounced Increase ◽

E Coli ◽

Amino Terminal ◽

Azo Reductase ◽

Anaerobic Reduction

ABSTRACT Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.

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Purification and characterization of Haemophilus influenzae pili, and their structural and serological relatedness to Escherichia coli P and mannose-sensitive pili.

Journal of Experimental Medicine ◽

10.1084/jem.161.1.145 ◽

1985 ◽

Vol 161 (1) ◽

pp. 145-159 ◽

Cited By ~ 13

Author(s):

N G Guerina ◽

S Langermann ◽

G K Schoolnik ◽

T W Kessler ◽

D A Goldmann

Keyword(s):

Haemophilus Influenzae ◽

Cross Reactivity ◽

Coli Strain ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Cell Agglutination ◽

E Coli ◽

Amino Terminal ◽

Multiple Copies

Haemophilus influenzae pili were purified, and their physical and serological properties were examined. The solution properties of the pili were determined, and then a purification scheme involving repeated cycles of precipitation and solubilization was developed. The purified pili from one type b isolate (A02) were found to consist of multiple copies of a 25,000 mol wt subunit. Amino-terminal sequence analysis of A02 pili was carried out to 40 amino acid residues, and a remarkable degree of sequence homology was found with E. coli P and mannose-sensitive (MS) pili (27.5 and 25% homology, respectively). Purified A02 pili were found to be highly immunogenic, and serological analysis by enzyme-linked immunosorbent assay and whole piliated cell agglutination revealed significant cross-reactivity between A02 pilus antiserum and the pili of seven other H. influenzae strains tested (heterologous titers = 2-100% of the homologous titer). Cross-reactivity was also observed between the H. influenzae pili (five of eight strains tested) and the P pili from E. coli strains HU849 and 3669; no cross-reactivity was detected with MS pili from E. coli strain H10407 and C94. The structural similarities between H. influenzae and E. coli P and MS pili suggest a common gene ancestry.

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Electron density, disorder and polymorphism: high-resolution diffraction studies of the highly polymorphic neuralgic drug carbamazepine

Acta Crystallographica Section B Structural Science Crystal Engineering and Materials ◽

10.1107/s2052520615019538 ◽

2016 ◽

Vol 72 (1) ◽

pp. 39-50 ◽

Cited By ~ 11

Author(s):

Ioana Sovago ◽

Matthias J. Gutmann ◽

Hans Martin Senn ◽

Lynne H. Thomas ◽

Chick C. Wilson ◽

...

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Diffraction Data ◽

Structural Formula ◽

Orthorhombic Crystal ◽

Monoclinic Crystal ◽

Orthorhombic Crystal Structure ◽

Space Group ◽

X Ray ◽

Monoclinic Crystal Structure

Analysis of neutron and high-resolution X-ray diffraction data on form (III) of carbamazepine at 100 K using the atoms in molecules (AIM) topological approach afforded excellent agreement between the experimental results and theoretical densities from the optimized gas-phase structure and from multipole modelling of static theoretical structure factors. The charge density analysis provides experimental confirmation of the partially localized π-bonding suggested by the conventional structural formula, but the evidence for any significant C—N π bonding is not strong. Hirshfeld atom refinement (HAR) gives H atom positional and anisotropic displacement parameters that agree very well with the neutron parameters. X-ray and neutron diffraction data on the dihydrate of carbemazepine strongly indicate a disordered orthorhombic crystal structure in the space groupCmca, rather than a monoclinic crystal structure in space groupP21/c. This disorder in the dihydrate structure has implications for both experimental and theoretical studies of polymorphism.

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Identification of Two New Proteins in Spermidine Nucleoids Isolated from Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.12.3842-3844.1999 ◽

1999 ◽

Vol 181 (12) ◽

pp. 3842-3844 ◽

Cited By ~ 12

Author(s):

Lizabeth D. Murphy ◽

Judah L. Rosner ◽

Steven B. Zimmerman ◽

Dominic Esposito

Keyword(s):

Escherichia Coli ◽

Functional Form ◽

Dnase I ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Form Analysis ◽

E Coli ◽

Amino Terminal

ABSTRACT The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC andyejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.

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Pilus-Mediated Adherence of Haemophilus influenzae to Human Respiratory Mucins

Infection and Immunity ◽

10.1128/iai.68.6.3362-3367.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3362-3367 ◽

Cited By ~ 33

Author(s):

Martin Kubiet ◽

Reuben Ramphal ◽

Allan Weber ◽

Arnold Smith

Keyword(s):

Escherichia Coli ◽

Haemophilus Influenzae ◽

Lung Disease ◽

Gene Cluster ◽

Otitis Media ◽

Structural Gene ◽

Chronic Lung Disease ◽

Type B ◽

E Coli

ABSTRACT Haemophilus influenzae, especially the nontypeable strains, are among the most common pathogens encountered in patients with chronic lung disease and otitis media. We and others have demonstrated that respiratory isolates of nontypeable H. influenzae bind to human mucins, but the mechanism of binding is not entirely clear. We have therefore examined the role of pili in the adherence of both type b and nontypeable H. influenzae to human respiratory mucins. We used isogenic H. influenzaestrains with a mutation in the structural gene for pilin (hifA), a laboratory H. influenzae strain transformed with a type b pilus gene cluster (from strain C54), antibodies raised against H. influenzae HifA, andEscherichia coli strains carrying a cloned type b pilus gene cluster (from strain AM30) in these studies. All bacteria lacking HifA or the pilus gene cluster had decreased adherence of piliatedH. influenzae to mucins, and Fab fragments of anti-HifA antibodies inhibited the adherence. E. coli strains carrying the cloned type b pilus gene cluster were six to seven times more adhesive than strains carrying the vector. The role of other putative adhesins was not examined and thus cannot be excluded, but these studies support a role for pili in the binding of H. influenzae to human respiratory mucins.

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The first step in sugar transport: crystal structure of the amino terminal domain of enzyme I of the E. coli PEP: sugar phosphotransferase system and a model of the phosphotransfer complex with HPr

Structure ◽

10.1016/s0969-2126(96)00092-5 ◽

1996 ◽

Vol 4 (7) ◽

pp. 861-872 ◽

Cited By ~ 84

Author(s):

D-I Liao ◽

E Silverton ◽

Y-J Seok ◽

BR Lee ◽

A Peterkofsky ◽

...

Keyword(s):

Crystal Structure ◽

Sugar Transport ◽

Phosphotransferase System ◽

E Coli ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain ◽

Enzyme I

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Expression of Alcaligenes eutrophusFlavohemoprotein and Engineered VitreoscillaHemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth ofEscherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.98-104.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 98-104 ◽

Cited By ~ 35

Author(s):

Alexander D. Frey ◽

James E. Bailey ◽

Pauli T. Kallio

Keyword(s):

Escherichia Coli ◽

Cell Density ◽

Product Formation ◽

Gene Encoding ◽

Final Cell Density ◽

E Coli ◽

Amino Terminal ◽

Positive Effects ◽

Cell Densities ◽

Carboxy Terminal

ABSTRACT Expression of the vhb gene encoding hemoglobin fromVitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant β-lactamase relative to the VHb-expressing control.

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