scholarly journals Slow-tight-binding inhibition of enoyl-acyl carrier protein reductase from Plasmodium falciparum by triclosan

2004 ◽  
Vol 381 (3) ◽  
pp. 719-724 ◽  
Author(s):  
Mili KAPOOR ◽  
C. Chandramouli REDDY ◽  
M. V. KRISHNASASTRY ◽  
Namita SUROLIA ◽  
Avadhesha SUROLIA
Keyword(s):  
Plasmodium Falciparum ◽  
Fatty Acid ◽  
Rate Constants ◽  
Enzyme Inhibitor ◽  
Acyl Carrier Protein ◽  
Tight Binding ◽  
Carrier Protein ◽  
Type I ◽  
Type Ii ◽  
Inhibitor Complex

Triclosan is a potent inhibitor of FabI (enoyl-ACP reductase, where ACP stands for acyl carrier protein), which catalyses the last step in a sequence of four reactions that is repeated many times with each elongation step in the type II fatty acid biosynthesis pathway. The malarial parasite Plasmodium falciparum also harbours the genes and is capable of synthesizing fatty acids by utilizing the enzymes of type II FAS (fatty acid synthase). The basic differences in the enzymes of type I FAS, present in humans, and type II FAS, present in Plasmodium, make the enzymes of this pathway a good target for antimalarials. The steady-state kinetics revealed time-dependent inhibition of FabI by triclosan, demonstrating that triclosan is a slow-tight-binding inhibitor of FabI. The inhibition followed a rapid equilibrium step to form a reversible enzyme–inhibitor complex (EI) that isomerizes to a second enzyme–inhibitor complex (EI*), which dissociates at a very slow rate. The rate constants for the isomerization of EI to EI* and the dissociation of EI* were 5.49×10−2 and 1×10−4 s−1 respectively. The Ki value for the formation of the EI complex was 53 nM and the overall inhibition constant Ki* was 96 pM. The results match well with the rate constants derived independently from fluorescence analysis of the interaction of FabI and triclosan, as well as those obtained by surface plasmon resonance studies [Kapoor, Mukhi, N. Surolia, Sugunda and A. Surolia (2004) Biochem. J. 381, 725–733].

scholarly journals Mutational analysis of the triclosan-binding region of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum

2004 ◽  
Vol 381 (3) ◽  
pp. 735-741 ◽  
Author(s):  
Mili KAPOOR ◽  
Jayashree GOPALAKRISHNAPAI ◽  
Namita SUROLIA ◽  
Avadhesha SUROLIA
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Affinity ◽  
Mutational Analysis ◽  
Acyl Carrier Protein ◽  
Striking Difference ◽  
Carrier Protein ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Binding Region

Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum, the human malaria-causing parasite, harbours the type II FAS; in contrast, its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P. falciparum ENR, have highlighted the features of ternary complex formation between the enzyme, triclosan and NAD+ [Suguna, A. Surolia and N. Surolia (2001) Biochem. Biophys. Res. Commun. 283, 224–228; Perozzo, Kuo, Sidhu, Valiyaveettil, Bittman, Jacobs, Fidock, and Sacchettini (2002) J. Biol. Chem. 277, 13106–13114; and Swarnamukhi, Kapoor, N. Surolia, A. Surolia and Suguna (2003) PDB1UH5]. To address the issue of the importance of the residues involved in strong specific and stoichiometric binding of triclosan to P. falciparum ENR, we mutated the following residues: Ala-217, Asn-218, Met-281, and Phe-368. The affinity of all the mutants was reduced for triclosan as compared with the wild-type enzyme to different extents. The most significant mutation was A217V, which led to a greater than 7000-fold decrease in the binding affinity for triclosan as compared with wild-type PfENR. A217G showed only 10-fold reduction in the binding affinity. Thus, these studies point out significant differences in the triclosan-binding region of the P. falciparum enzyme from those of its bacterial counterparts.

scholarly journals A Mammalian Type I Fatty Acid Synthase Acyl Carrier Protein Domain Does Not Sequester Acyl Chains

2007 ◽  
Vol 283 (1) ◽  
pp. 518-528 ◽  
Author(s):  
Eliza Ploskoń ◽  
Christopher J. Arthur ◽  
Simon E. Evans ◽  
Christopher Williams ◽  
John Crosby ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domain ◽  
Carrier Protein ◽  
Type I ◽  
Acyl Chains

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

scholarly journals Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

1996 ◽  
Vol 40 (12) ◽  
pp. 2813-2819 ◽  
Author(s):  
R A Slayden ◽  
R E Lee ◽  
J W Armour ◽  
A M Cooper ◽  
I M Orme ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Type I ◽  
Dependent Manner ◽  
Fas Ii

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

scholarly journals 1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

2004 ◽  
Vol 48 (8) ◽  
pp. 3093-3102 ◽  
Author(s):  
Xin He ◽  
Anne McElwee Reeve ◽  
Umesh R. Desai ◽  
Glen E. Kellogg ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Enzyme Inhibitor ◽  
Acyl Carrier Protein ◽  
Phenyl Group ◽  
Initial Step ◽  
Structural Features ◽  
Inhibitor Complex

ABSTRACT The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered.

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 Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

2016 ◽  
Vol 84 (12) ◽  
pp. 3597-3607 ◽  
Author(s):  
Jiangwei Yao ◽  
Megan E. Ericson ◽  
Matthew W. Frank ◽  
Charles O. Rock
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Type Ii ◽  
Intracellular Growth ◽  
Content Type ◽  
Bacterial Fatty Acid

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogenListeria monocytogenesencode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype ofEscherichia colistrain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate ofL. monocytogenesin laboratory medium. Robust exogenous fatty acid incorporation was not detected inL. monocytogenesunless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth ofL. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth ofL. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth ofL. monocytogenes.

scholarly journals Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme

2000 ◽  
Vol 345 (1) ◽  
pp. 153 ◽  
Author(s):  
Amine ABBADI ◽  
Monika BRUMMEL ◽  
Burkhardt S. SCHÜTT ◽  
Mary B. SLABAUGH ◽  
Ricardo SCHUCH ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Reaction Mechanism ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Type Ii ◽  
Condensing Enzyme

scholarly journals The Structure of (3R)-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) fromPseudomonas aeruginosa

2004 ◽  
Vol 279 (50) ◽  
pp. 52593-52602 ◽  
Author(s):  
Matthew S. Kimber ◽  
Fernando Martin ◽  
Yingjie Lu ◽  
Simon Houston ◽  
Masoud Vedadi ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Isomerization Reaction ◽  
Antibacterial Drug ◽  
Carrier Protein ◽  
Type Ii ◽  
Active Site Residues ◽  
Acid Biosynthesis

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by β-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP totrans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization oftrans-2- tocis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogenPseudomonas aeruginosaat 2.5 Å of resolution.PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

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