Using native MS to identify homotropic allosteric effects of the coenzymes of enoyl-ACP reductase from E. coli (FabI)

AbstractWe report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize two-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 16 fold and 100 fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions.HighlightsDecreases in NADPH pools lead to increased NADPH fluxesPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo.Dynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH fluxXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock.

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Characterization of protein-ligand binding interactions of enoyl-ACP reductase (FabI) by native MS reveals allosteric effects of coenzymes and the inhibitor triclosan

10.1101/2019.12.30.891283 ◽

2019 ◽

Author(s):

P. Matthew Joyner ◽

Denise P. Tran ◽

Muhammad A. Zenaidee ◽

Joseph A. Loo

Keyword(s):

Fatty Acid Synthase ◽

Antibacterial Drug ◽

Allosteric Effect ◽

The Past ◽

Protein Ligand Interactions ◽

Native Ms ◽

Ligand Interactions ◽

Enoyl Acp Reductase ◽

The Subject

AbstractThe enzyme enoyl-ACP reductase (also called FabI in bacteria) is an essential member of the fatty acid synthase II pathway in plants and bacteria. This enzyme is the target of the antibacterial drug triclosan and has been the subject of extensive studies for the past 20 years. Despite the large number of reports describing the biochemistry of this enzyme, there have been no studies that provided direct observation of the protein and its various ligands. Here we describe the use of native MS to characterize the protein-ligand interactions of FabI with its coenzymes NAD+ and NADH and with the inhibitor triclosan. Measurements of the gas-phase affinities of the enzyme for these ligands yielded values that are in close agreement with solution-phase affinity measurements. Additionally, FabI is a homotetramer and we were able to measure the affinity of each subunit for each coenzyme, which revealed that both coenzymes exhibit a positive homotropic allosteric effect. An allosteric effect was also observed in association with the inhibitor triclosan. These observations provide new insights into this well-studied enzyme and suggest that there may still be gaps in the existing mechanistic models that explain FabI inhibition.

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Synthesis and Bacteriostatic Activities of Bis(thiourea) Derivatives with Variable Chain Length

Journal of Chemistry ◽

10.1155/2016/2739832 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Ainaa Nadiah Abd Halim ◽

Zainab Ngaini

Keyword(s):

Chain Length ◽

Alkyl Chain ◽

Binding Free Energy ◽

Kinetic Method ◽

Antibacterial Properties ◽

Thiourea Derivatives ◽

E Coli ◽

Excellent Activity ◽

Enoyl Acp Reductase ◽

Long Alkyl Chain

A series of 1,4-bis(decoxyphenyl)carbamothioyl-terephthalamide derivatives was successfully synthesised by reaction of benzene-1,4-dicarbonyl isothiocyanate intermediates with long alkyl chain. The alkylation was performed via Williamson etherification of 4-acetamidophenol with bromoalkanes. The synthesised bis(thiourea) derivatives differed in the chain length, CnH2n+1, wheren=10, 12, and 14. The structures of all compounds were characterised by elemental CHN analysis, IR,1H, and13C NMR spectroscopies. Bacteriostatic activities of bis(thiourea derivatives which consisted of two folds of N-H, C=O, and C=S and long alkyl chain substituents were carried out against Gram-negative bacteria (Escherichia coli, ATCC 25922) via turbidimetric kinetic method. Bis(thiourea) derivatives withn=10andn=12displayed excellent activity againstE. coliwith MIC of 135 µg/mL and 145 µg/mL, respectively, while bis(thiourea) derivatives withn=14acted as cutoff point with no antibacterial properties. Similar trend was observed in binding affinity to the active site of enoyl ACP reductase (FabI), which demonstrated binding free energy of-5.3 Kcal/mol and-4.9and-4.8 Kcal/mol, respectively.

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ChemInform Abstract: Spiro-Naphthyridinone Piperidines as Inhibitors of S. aureus and E. coli Enoyl-ACP Reductase (FabI).

ChemInform ◽

10.1002/chin.201005144 ◽

2010 ◽

Vol 41 (5) ◽

Author(s):

Peter B. Sampson ◽

et al. et al.

Keyword(s):

E Coli ◽

Enoyl Acp Reductase

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ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000441640 ◽

2015 ◽

Vol 25 (6) ◽

pp. 394-402 ◽

Cited By ~ 1

Author(s):

Taylor L. Fischer ◽

Robert J. White ◽

Katherine F.K. Mares ◽

Devin E. Molnau ◽

Justin J. Donato

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Wild Type ◽

E Coli ◽

Enoyl Acp Reductase ◽

Selection For

Background/Aims: We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. Methods: ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. Results: Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. Conclusion: ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

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Triclosan Resistance of Pseudomonas aeruginosa PAO1 Is Due to FabV, a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01152-09 ◽

2009 ◽

Vol 54 (2) ◽

pp. 689-698 ◽

Cited By ~ 107

Author(s):

Lei Zhu ◽

Jinshui Lin ◽

Jincheng Ma ◽

John E. Cronan ◽

Haihong Wang

Keyword(s):

Pseudomonas Aeruginosa ◽

Mutant Strain ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Carrier Protein ◽

Active Efflux ◽

E Coli ◽

Genes Encoding ◽

Enoyl Acp Reductase ◽

Triclosan Resistance

ABSTRACT Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacteria are resistant to triclosan, because they encode triclosan-resistant enoyl-ACP reductase isozymes. In contrast, the triclosan resistance of Pseudomonas aeruginosa PAO1 has been attributed to active efflux of the compound (R. Chuanchuen, R. R. Karkhoff-Schweizer, and H. P. Schweizer, Am. J. Infect. Control 31:124-127, 2003). We report that P. aeruginosa contains two enoyl-ACP reductase isozymes, the previously characterized FabI homologue plus a homologue of FabV, a triclosan-resistant enoyl-ACP reductase recently demonstrated in Vibrio cholerae. By deletion of the genes encoding P. aeruginosa FabI and FabV, we demonstrated that FabV confers triclosan resistance on P. aeruginosa. Upon deletion of the fabV gene, the mutant strain became extremely sensitive to triclosan (>2,000-fold more sensitive than the wild-type strain), whereas the mutant strain lacking FabI remained completely resistant to the biocide.

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