scholarly journals Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Raees Khan ◽  
Amir Zeb ◽  
Nazish Roy ◽  
Roniya Thapa Magar ◽  
Hyo Jeong Kim ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Carrier Protein ◽  
Content Type ◽  
Triclosan Resistance ◽  
Bacterial Type

ABSTRACTEnoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found inEpsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the generaHelicobacterandCampylobacter. Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenicEpsilonproteobacteria.

scholarly journals Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Raees Khan ◽  
Amir Zeb ◽  
Kihyuck Choi ◽  
Gihwan Lee ◽  
Keun Woo Lee ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Fatty Acid Biosynthesis ◽  
Catalytic Domain ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Carrier Protein ◽  
The Novel ◽  
Triclosan Resistance ◽  
Structural Insights

Abstract Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

The role of β-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower

Planta ◽  
2010 ◽  
Vol 231 (6) ◽  
pp. 1277-1289 ◽  
Author(s):  
Damián González-Mellado ◽  
Penny von Wettstein-Knowles ◽  
Rafael Garcés ◽  
Enrique Martínez-Force
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Acid Biosynthesis

Triclosan Resistance in Francisella tularensis: A Site-Directed Mutagenesis Study of the FabI Enzyme

2015 ◽  
Vol 8 (1) ◽  
Author(s):  
N. Silas ◽  
R. Demissie ◽  
L.W.M. Fung
Keyword(s):  
Fatty Acid ◽  
Francisella Tularensis ◽  
Bacterial Resistance ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Enzymatic Reaction ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Bacterial Fatty Acid ◽  
Triclosan Resistance

An NADH-dependent enoyl-acyl carrier protein reductase, FabI, catalyzes the final step of bacterial fatty acid biosynthesis, reducing the double bond of trans-2-enoyl-ACP to a single bond forming acyl-ACP. Given its importance in bacterial fatty acid synthesis, FabI has become a recognized drug target. Triclosan, a diphenyl ether, targets the FabI, inhibits its activity, and stops bacterial growth. However, as a consequence of triclosan's popularity, and thus its overuse, bacterial resistance to triclosan has been reported. The mutation G93V in Escherichia coli (E. coli) FabI allows E. coli to resist the action of triclosan. We have identified the equivalent residue of G93 in Francisella tularensis FabI (ftFabI) as A92, and prepared a mutant A92V. E. coli cells, transformed with a plasmid containing the ftFabI-A92V gene, were grown, and the gene was overexpressed. From two growths (6 G of cells), 62 mG of protein, with a histidine tag, at a purity of 85% were obtained. Enzymatic activity was assayed by monitoring the absorbance of NADH at 340 nm. In the presence of triclosan, the wild-type protein was almost completely inhibited after NADH was converted to NAD$^+$ in the enzymatic reaction; however the A92V mutant exhibited similar activity with and without triclosan, demonstrating that triclosan resistance may also develop in Francisella tularensis.

scholarly journals The Burkholderia pseudomallei Enoyl-Acyl Carrier Protein Reductase FabI1 Is Essential forIn VivoGrowth and Is the Target of a Novel Chemotherapeutic with Efficacy

2013 ◽  
Vol 58 (2) ◽  
pp. 931-935 ◽  
Author(s):  
Jason E. Cummings ◽  
Luke C. Kingry ◽  
Drew A. Rholl ◽  
Herbert P. Schweizer ◽  
Peter J. Tonge ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Specific Inhibitor ◽  
Carrier Protein ◽  
Wild Type ◽  
Content Type ◽  
Fatty Acid Biosynthesis Pathway ◽  
Enoyl Acp Reductase

ABSTRACTThe bacterial fatty acid biosynthesis pathway is a validated target for the development of novel chemotherapeutics. However, sinceBurkholderia pseudomalleicarries genes that encode both FabI and FabV enoyl-acyl carrier protein (ACP) reductase homologues, the enoyl-ACP reductase that is essential forin vivogrowth needs to be defined so that the correct drug target can be chosen for development. Accordingly, ΔfabI1, ΔfabI2, and ΔfabVknockout strains were constructed and tested in a mouse model of infection. Mice infected with a ΔfabI1strain did not show signs of morbidity, mortality, or dissemination after 30 days of infection compared to the wild-type and ΔfabI2and ΔfabVmutant strains that had times to mortality of 60 to 84 h. Although signs of morbidity and mortality of ΔfabI2and ΔfabVstrains were not significantly different from those of the wild-type strain, a slight delay was observed. A FabI1-specific inhibitor was used to confirm that inhibition of FabI1 results in reduced bacterial burden and efficacy in an acuteB. pseudomalleimurine model of infection. This work establishes that FabI1 is required for growth ofBurkholderia pseudomalleiin vivoand is a potential molecular target for drug development.

scholarly journals A Conserved Histidine Is Essential for Glycerolipid Acyltransferase Catalysis

1998 ◽  
Vol 180 (6) ◽  
pp. 1425-1430 ◽  
Author(s):  
Richard J. Heath ◽  
Charles O. Rock
Keyword(s):  
Aspartic Acid ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Carrier Protein ◽  
Synthetase Activity ◽  
Acyltransferase Activity ◽  
Acyl Acceptor ◽  
Aspartic Acid Residue ◽  
Membrane Bound

ABSTRACT Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) ofEscherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.

scholarly journals The Two Functional Enoyl-Acyl Carrier Protein Reductases of Enterococcus faecalis Do Not Mediate Triclosan Resistance

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lei Zhu ◽  
Hongkai Bi ◽  
Jincheng Ma ◽  
Zhe Hu ◽  
Wenbin Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Enterococcus Faecalis ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Content Type ◽  
Enoyl Acp Reductase ◽  
Triclosan Resistance

ABSTRACTEnoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. TheEnterococcus faecalisgenome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of anEscherichia colifabItemperature-sensitive mutant strain under nonpermissive conditions.In vitroassays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression ofE. faecalis fabKconfers toE. colihigh levels of resistance to the antimicrobial triclosan, deletion offabKfrom theE. faecalisgenome showed that FabK does not play a detectable role in the inherent triclosan resistance ofE. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion offabKgrow normally without fatty acid supplementation, whereasfabIdeletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs.IMPORTANCEThe finding that exogenous fatty acids support growth ofE. faecalisstrains defective in fatty acid synthesis indicates that inhibitors of fatty acid synthesis are ineffective in counteringE. faecalisinfections because host serum fatty acids support growth of the bacterium.

The Role of Tandem Acyl Carrier Protein Domains in Polyunsaturated Fatty Acid Biosynthesis

2008 ◽  
Vol 130 (20) ◽  
pp. 6336-6337 ◽  
Author(s):  
Hui Jiang ◽  
Ross Zirkle ◽  
James G. Metz ◽  
Lisa Braun ◽  
Leslie Richter ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Polyunsaturated Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Protein Domains ◽  
Carrier Protein ◽  
Acid Biosynthesis

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.

scholarly journals Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

2011 ◽  
Vol 78 (5) ◽  
pp. 1563-1573 ◽  
Author(s):  
Juanli Cheng ◽  
Jincheng Ma ◽  
Jinshui Lin ◽  
Zhen-Chuan Fan ◽  
John E. Cronan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Ralstonia Solanacearum ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Phytopathogenic Bacterium ◽  
Content Type ◽  
Long Chain

ABSTRACTRalstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes ofR. solanacearumshowed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) andcis-vaccenic (C18:1) acids, little is known regardingR. solanacearumfatty acid synthesis. TheR. solanacearumGMI1000 genome is unusual in that it contains four genes (fabF1,fabF2,fabF3, andfabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes,fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles inR. solanacearumfatty acid synthesis. Mutant strains lackingfabF1are nonviable, and thus, FabF1 is essential forR. solanacearumfatty acid biosynthesis. Moreover,R. solanacearumFabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I.

scholarly journals Activity Mapping the Acyl Carrier Protein - Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis

2020 ◽  
Author(s):  
Jeffrey T. Mindrebo ◽  
Laetitia E. Misson ◽  
Caitlin Johnson ◽  
Joseph P. Noel ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Chain Extension ◽  
Carrier Protein ◽  
Type Ii ◽  
Multiple Partner ◽  
Carbon Carbon Bond

ABSTRACTElongating ketosynthases (KSs) catalyze carbon-carbon bond forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution detail of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region delete variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of entire regions significantly compromising activity. Structure and sequence analysis of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.

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