scholarly journals Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

F1000Research ◽  
2022 ◽  
Vol 10 ◽  
pp. 1102
Author(s):  
Vladyslav Yadrykhins'ky ◽  
Charis Georgiou ◽  
Ruth Brenk
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Aeruginosa ◽  
Fatty Acid ◽  
Binding Site ◽  
Acyl Carrier Protein ◽  
Size Exclusion ◽  
Carrier Protein ◽  
Fatty Acid Binding ◽  
Efficient System ◽  
Tev Protease

Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl-enzyme intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB (PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E. coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Binding site comparison between PaFabB and PaFabF revealed a conserved malonyl binding site but differences in the fatty acid binding channel. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.

scholarly journals Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 1102
Author(s):  
Vladyslav Yadrykhins'ky ◽  
Charis Georgiou ◽  
Ruth Brenk
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Aeruginosa ◽  
Acyl Carrier Protein ◽  
Recombinant Expression ◽  
Acid Synthesis ◽  
Size Exclusion ◽  
Carrier Protein ◽  
Efficient System ◽  
Tev Protease ◽  
Exclusion Chromatography

Background: FabB (3-oxoacyl-[acyl-carrier-protein] synthase 1) is part of the fatty acid synthesis II pathway found in bacteria and a potential target for antibiotics. The enzyme catalyses the Claisen condensation of malonyl-ACP (acyl carrier protein) with acyl-ACP via an acyl intermediate. Here, we report the crystal structure of the intermediate-mimicking Pseudomonas aeruginosa FabB (PaFabB) C161A variant. Methods: His-tagged PaFabB C161A was expressed in E.coli Rosetta DE3 pLysS cells, cleaved by TEV protease and purified using affinity and size exclusion chromatography. Commercial screens were used to identify suitable crystallization conditions which were subsequently improved to obtain well diffracting crystals. Results: We developed a robust and efficient system for recombinant expression of PaFabB C161A. Conditions to obtain well diffracting crystals were established. The crystal structure of PaFabB C161A was solved by molecular replacement at 1.3 Å resolution. Conclusions: The PaFabB C161A crystal structure can be used as a template to facilitate the design of FabB inhibitors.

scholarly journals RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids in Pseudomonas aeruginosa

2008 ◽  
Vol 190 (9) ◽  
pp. 3147-3154 ◽  
Author(s):  
Kun Zhu ◽  
Charles O. Rock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Molecular Ruler ◽  
Necessary And Sufficient

ABSTRACT Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of β-hydroxydecanoyl-β-hydroxydecanoate from two molecules of β-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily β-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for β-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.

scholarly journals Kinetic, inhibition and structural studies on 3-oxoacyl-ACP reductase from Plasmodium falciparum, a key enzyme in fatty acid biosynthesis

2005 ◽  
Vol 393 (2) ◽  
pp. 447-457 ◽  
Author(s):  
Sasala R. Wickramasinghe ◽  
Kirstine A. Inglis ◽  
Jonathan E. Urch ◽  
Sylke Müller ◽  
Daan M. F. van Aalten ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Plasmodium Falciparum ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Competitive Inhibition ◽  
Acyl Carrier Protein ◽  
Antimalarial Activity ◽  
Kinetic Properties ◽  
Carrier Protein ◽  
Acid Biosynthesis

Type II fatty acid biosynthesis represents an attractive target for the discovery of new antimalarial drugs. Previous studies have identified malarial ENR (enoyl acyl-carrier-protein reductase, or FabI) as the target for the antiseptic triclosan. In the present paper, we report the biochemical properties and 1.5 Å (1 Å=0.1 nm) crystal structure of OAR (3-oxoacyl acyl-carrier-protein reductase, or FabG), the second reductive step in fatty acid biosynthesis and its inhibition by hexachlorophene. Under optimal conditions of pH and ionic strength, Plasmodium falciparum OAR displays kinetic properties similar to those of OAR from bacteria or plants. Activity with NADH is <3% of that with NADPH. Fluorescence enhancement studies indicate that NADPH can bind to the free enzyme, consistent with kinetic and product inhibition studies suggesting a steady-state ordered mechanism. The crystal structure reveals a tetramer with a sulphate ion bound in the cofactor-binding site such that the side chains of the catalytic triad of serine, tyrosine and lysine are aligned in an active conformation, as previously observed in the Escherichia coli OAR–NADP+ complex. A cluster of positively charged residues is positioned at the entrance to the active site, consistent with the proposed recognition site for the physiological substrate (3-oxoacyl-acyl-carrier protein) in E. coli OAR. The antibacterial and anthelminthic agent hexachlorophene is a potent inhibitor of OAR (IC50 2.05 μM) displaying non-linear competitive inhibition with respect to NADPH. Hexachlorophene (EC50 6.2 μM) and analogues such as bithionol also have antimalarial activity in vitro, suggesting they might be useful leads for further development.

scholarly journals Purification, Characterization, and Identification of Novel Inhibitors of the β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from Staphylococcus aureus

2002 ◽  
Vol 46 (5) ◽  
pp. 1310-1318 ◽  
Author(s):  
Xin He ◽  
Kevin A. Reynolds
Keyword(s):  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Genomic Sequence ◽  
Acyl Carrier Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Carrier Protein

ABSTRACT Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent Km for malonyl-ACP was 1.76 ± 0.40 μM, and the enzyme was active with acetyl-CoA (k cat, 16.18 min−1; Km , 6.18 ± 0.9 μM), butyryl-CoA (k cat, 42.90 min−1; Km , 2.32 ± 0.12 μM), and isobutyryl-CoA (k cat, 98.0 min−1; Km , 0.32 ± 0.04 μM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 μM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 ± 0.10 μM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 ± 0.08 μM).

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