Antibacterial Strategy against <i>H. pylori</i>: Inhibition of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis

Joshi, Sumedh; Fedoseyenko, Dmytro; Mahanta, Nilkamal; Ducati, Rodrigo G.; Feng, Mu; Schramm, Vern L.; Begley, Tadhg P.

doi:10.1021/acsmedchemlett.8b00649

Cited by 16 publications

(14 citation statements)

References 33 publications

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“…Such a drug design strategy has been successfully applied to inhibit critical metabolic pathways in MpnE [14] and purine-nucleotide phosphorylase [15]. Inhibitors created from mechanistic insights of TetX would then be used in conjugation with existing tetracyclines that are currently on the market to treat infectious disease.…”

Section: Discussionmentioning

confidence: 99%

An Improved Protocol for the Expression and Purification of Tetracycline Monooxgenase: An Enzyme Involved in Antibiotic Resistance

Francis¹

2020

AABSc

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Tetracyclines are an important group of antibiotics used in agricultural and clinical settings. They function by binding to the 30S subunit of the prokaryotic ribosome to effectively halt translation. The emergence of tetracycline resistant strains of pathogenic bacteria has threatened the continued use of the drug. One mechanism of tetracycline resistance is the degradation of the drug by the flavin dependent enzyme tetracycline monooxygenase (TetX). Only a limited number of studies of the enzyme have been conducted to date due to the limited amounts of pure protein that can be obtained from current protocols (~6 mg/L of cell culture). The current report describes the development of optimized expression and purification protocols to obtain larger amounts of pure, soluble enzyme suitable for detailed kinetic studies to deduce its chemical mechanism. During the initial phase of this work the pET22a+ plasmid with the gene encoding for TetX was used to transform either DH5α or Rosetta (DE3) pLysS Escherichia coli cells. Expression trials of these cell lines in which both the temperature and times of incubation with isopropyl β-D-1-thiogalactopyrandoside were then carried out. Of the conditions tested, optimal expression of TetX was found with a 20 h induction period at 30 °C using E. coli DH5α cells. Ammonium sulfate precipitation trials were then conducted where it was concluded that a 20 fold purification of the enzyme is achieved through treatment with 40% saturation of salt and collection of the supernatant after centrifugation. A nickel affinity chromatographic protocol was also developed, which purified the enzyme to high levels as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the specific activity of the purified sample. The expression and purification protocol developed here resulted in ~36 mg/L of cell culture, which is a 6-fold improvement over published protocols.

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Section: Discussionmentioning

confidence: 99%

An Improved Protocol for the Expression and Purification of Tetracycline Monooxgenase: An Enzyme Involved in Antibiotic Resistance

Francis¹

2020

AABSc

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“…A methylene analogue of DHC ( Figure 10 B) was shown to reversibly inhibit MqnE with the K i of 3.1 ± 0.1 μM. 89 The absence of irreversible inhibition was established by restoration of the activity after the removal of the inhibitor. Details of the mechanism of inhibition by this methylene analogue have not been reported.…”

Section: Translating the Mechanistic And Structural Understanding To ...mentioning

confidence: 99%

Resolving the Multidecade-Long Mystery in MoaA Radical SAM Enzyme Reveals New Opportunities to Tackle Human Health Problems

Yokoyama

Pang

2021

ACS Bio Med Chem Au

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MoaA is one of the most conserved radical S -adenosyl- l -methionine (SAM) enzymes, and is found in most organisms in all three kingdoms of life. MoaA contributes to the biosynthesis of molybdenum cofactor (Moco), a redox enzyme cofactor used in various enzymes such as purine and sulfur catabolism in humans and anaerobic respiration in bacteria. Unlike many other cofactors, in most organisms, Moco cannot be taken up as a nutrient and requires de novo biosynthesis. Consequently, Moco biosynthesis has been linked to several human health problems, such as human Moco deficiency disease and bacterial infections. Despite the medical and biological significance, the biosynthetic mechanism of Moco’s characteristic pyranopterin structure remained elusive for more than two decades. This transformation requires the actions of the MoaA radical SAM enzyme and another protein, MoaC. Recently, MoaA and MoaC functions were elucidated as a radical SAM GTP 3′,8-cyclase and cyclic pyranopterin monophosphate (cPMP) synthase, respectively. This finding resolved the key mystery in the field and revealed new opportunities in studying the enzymology and chemical biology of MoaA and MoaC to elucidate novel mechanisms in enzyme catalysis or to address unsolved questions in Moco-related human health problems. Here, we summarize the recent progress in the functional and mechanistic studies of MoaA and MoaC and discuss the field’s future directions.

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“…Some of the tested inhibitors were able to penetrate the H. pylori cell wall as they inhibited the pathogen growth at low concentration (Wang et al 2012 ; Wang et al 2015 ; Harijan et al 2019 ). Aminofutalosine synthase (MqnE) is another enzyme of the futalosine pathway of menaquinone biosynthesis indicated as a potential target for new antibiotics (Joshi et al 2019 ).…”

Section: Searching For New Drug Targets To Combat H Pylorimentioning

confidence: 99%

Helicobacter pylori treatment in the post-antibiotics era—searching for new drug targets

Roszczenko-Jasińska

Wojtyś

Jagusztyn-Krynicka

2020

Appl Microbiol Biotechnol

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Helicobacter pylori, a member of Epsilonproteobacteria, is a Gram-negative microaerophilic bacterium that colonizes gastric mucosa of about 50% of the human population. Although most infections caused by H. pylori are asymptomatic, the microorganism is strongly associated with serious diseases of the upper gastrointestinal tract such as chronic gastritis, peptic ulcer, duodenal ulcer, and gastric cancer, and it is classified as a group I carcinogen. The prevalence of H. pylori infections varies worldwide. The H. pylori genotype, host gene polymorphisms, and environmental factors determine the type of induced disease. Currently, the most common therapy to treat H. pylori is the first line clarithromycin–based triple therapy or a quadruple therapy replacing clarithromycin with new antibiotics. Despite the enormous recent effort to introduce new therapeutic regimens to combat this pathogen, treatment for H. pylori still fails in more than 20% of patients, mainly due to the increased prevalence of antibiotic resistant strains. In this review we present recent progress aimed at designing new anti-H. pylori strategies to combat this pathogen. Some novel therapeutic regimens will potentially be used as an extra constituent of antibiotic therapy, and others may replace current antibiotic treatments. Key points • Attempts to improve eradication rate of H. pylori infection. • Searching for new drug targets in anti-Helicobacter therapies.

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Antibacterial Strategy against H. pylori: Inhibition of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis

Cited by 16 publications

References 33 publications

An Improved Protocol for the Expression and Purification of Tetracycline Monooxgenase: An Enzyme Involved in Antibiotic Resistance

An Improved Protocol for the Expression and Purification of Tetracycline Monooxgenase: An Enzyme Involved in Antibiotic Resistance

Resolving the Multidecade-Long Mystery in MoaA Radical SAM Enzyme Reveals New Opportunities to Tackle Human Health Problems

Helicobacter pylori treatment in the post-antibiotics era—searching for new drug targets

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