An in vivo pathway for disulfide bond isomerization in Escherichia coli

Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.

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Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0403003101 ◽

2004 ◽

Vol 101 (27) ◽

pp. 10018-10023 ◽

Cited By ~ 34

Author(s):

L. Segatori ◽

P. J. Paukstelis ◽

H. F. Gilbert ◽

G. Georgiou

Keyword(s):

Escherichia Coli ◽

Disulfide Bond ◽

Protein Oxidation ◽

Disulfide Bond Isomerization ◽

Competing Pathways

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Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ70 Binding

Journal of Bacteriology ◽

10.1128/jb.184.10.2634-2641.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2634-2641 ◽

Cited By ~ 11

Author(s):

Larry C. Anthony ◽

Alan A. Dombkowski ◽

Richard R. Burgess

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Disulfide Bond ◽

Conformational Changes ◽

Coiled Coil ◽

Mrna Synthesis ◽

Promoter Sequences ◽

Coiled Coil Region

ABSTRACT RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ70. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ70. We also show that the locked coiled-coil is capable of inducing a conformational change within σ70 that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ70 or upon recognition of the −10 nontemplate strand of DNA.

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Безопасность соединений из группы терпенов ментанового ряда in vitro и in vivo

Экспериментальная и клиническая фармакология ◽

10.30906/0869-2092-2020-83-4-24-30 ◽

2020 ◽

Vol 83 (4) ◽

pp. 24-30

Author(s):

Ирина Владимировна Акулина ◽

Светлана Ивановна Павлова ◽

Ирина Семеновна Степаненко ◽

Назира Сунагатовна Карамова ◽

Александр Владиславович Сергеев ◽

...

Keyword(s):

Escherichia Coli

Проведено токсикологическое исследование соединений с антибактериальными свойствами из группы терпенов ментанового ряда в условиях in vitro и in vivo: лимонена (B34), его производного (+)-1,2-оксида лимонена (B60) и серосодержащего монотерпенового соединения 2-(1’-гидрокси-4’-изопренил-1’-метилциклогексил-2’-тио)метилэтаноата (B65). В условиях in vitro (культура опухолевых клеток HeLa) изучаемые монотерпены в диапазоне концентраций 2 – 200 мкг/мл обладали цитотоксичностью. Ингибирующая концентрация (ИК50) для B34 составила 231 (167 – 295) мкг/мл, для B60 – 181 (105 – 257) мкг/мл, ИК50 B65 – 229 (150 – 308) мкг/мл. Исследование генотоксичности показало, что B34 и B65 в диапазоне концентраций 50 – 1000 мкг/мл не индуцируют SOS мутагенез в клетках Escherichia coli PQ37, тогда как B60 в концентрациях 500 и 1000 мкг/мл проявляет генотоксичность. In vivo в остром эксперименте на беспородных мышах установлена низкая токсичность B34 и его производных при различных путях введения. Наименьший показатель острой токсичности имеет B65, в связи с чем дополнительно на крысах проведено изучение его хронической токсичности. Ежедневное внутрижелудочное введение B65 в разовых дозах, составляющих 1/10 и 1/20 ЛД50 (1000 мг/кг и 500 мг/кг), в течение 1 мес не вызывало гибели животных, значимых нарушений общего состояния, изменения динамики массы тела, морфопатологических изменений. Внутрижелудочное введение B65 крысам в высокой токсической дозе 2000 мг/кг (1/5 ЛД50) в течение месяца вызывает патоморфологические изменения структуры печени.

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Faculty Opinions recommendation of In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014585.193921 ◽

2003 ◽

Author(s):

Jurgen Soll

Keyword(s):

Escherichia Coli ◽

Outer Membrane

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Faculty Opinions recommendation of An altered-specificity DNA-binding mutant of Escherichia coli sigma70 facilitates the analysis of sigma70 function in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026042.322916 ◽

2005 ◽

Author(s):

Stephen Busby

Keyword(s):

Escherichia Coli ◽

Dna Binding

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Garlic Extract (Allium sativum L.) Effectively Inhibits Staphylococcus aureus and Escherichia coli by Invitro Test

Tropical Health and Medical Research ◽

10.35916/thmr.v0i0.22 ◽

2020 ◽

Vol 2 (2) ◽

pp. 61-68

Author(s):

Agnina Listya Anggraini ◽

Ratih Dewi Dwiyanti ◽

Anny Thuraidah

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Bacterial Infections ◽

Allium Sativum ◽

Antibacterial Properties ◽

Herbal Medicines ◽

Garlic Extract ◽

Dilution Method ◽

Initial Stage

Infection is a disease caused by the presence of pathogenic microbes, including Staphylococcus aureus and Escherichia coli. Garlic (Allium sativum L.) has chemical contents such as allicin, alkaloids, flavonoids, saponins, tannins, and steroids, which can function as an antibacterial against Staphylococcus aureus and Escherichia coli. This study aims to determine the antibacterial properties of garlic extract powder against Staphylococcus aureus and Escherichia coli. This research is the initial stage of the development of herbal medicines to treat Staphylococcus aureus and Escherichia coli infections. The antibacterial activity test was carried out by the liquid dilution method. The concentrations used were 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL and 70 mg/mL. The results showed that the Minimum Inhibitory Concentration (MIC) against Staphylococcus aureus and Escherichia coli was 40 mg/mL and 50 mg / mL. Minimum Bactericidal Concentration (MBC) results for Staphylococcus aureus and Escherichia coli are 50 mg/mL and 70 mg/mL. Based on the Simple Linear Regression test, the R2 value of Staphylococcus aureus and Escherichia coli is 0.545 and 0.785, so it can be concluded that there is an effect of garlic extract powder on the growth of Staphylococcus aureus and Escherichia coli by 54.5% and 78.5%. Garlic (Allium sativum L.) extract powder has potential as herbal medicine against bacterial infections but requires further research to determine its effect in vivo.

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