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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00648-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 3

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.

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

The Journal of Undergraduate Research at the University of Illinois at Chicago ◽

10.5210/jur.v8i1.7531 ◽

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m408105200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52593-52602 ◽

Cited By ~ 91

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

10.1101/2020.07.09.196451 ◽

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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Structural analysis and interaction studies of acyl-carrier protein (acpP) of Staphylococcus aureus, an extraordinarily thermally stable protein

Biological Chemistry ◽

10.1515/hsz-2016-0185 ◽

2017 ◽

Vol 398 (1) ◽

pp. 125-133 ◽

Cited By ~ 3

Author(s):

Kathrin Volk ◽

Sven D. Breunig ◽

Raphaela Rid ◽

Julia Herzog ◽

Maria Bräuer ◽

...

Keyword(s):

Escherichia Coli ◽

Heat Treatment ◽

Staphylococcus Aureus ◽

Fatty Acid ◽

Pathogenic Bacteria ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Carrier Protein ◽

Acid Biosynthesis ◽

Acta Crystallogr

Abstract Acyl-carrier-protein (acpP) is an essential protein in fatty acid biosynthesis of Staphylococcus aureus [Cronan, J.E. and Thomas, J. (2009). Complex enzymes in microbial natural product biosynthesis, part B: polyketides, aminocoumarins and carbohydrates. Method. Enzymol. 459, 395–433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359–1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4′-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959–968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270, 24658–24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359–1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649–662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [14C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.

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Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913021859 ◽

2013 ◽

Vol 69 (12) ◽

pp. 2340-2352 ◽

Cited By ~ 8

Author(s):

Karine Moncoq ◽

Leslie Regad ◽

Stéphane Mann ◽

Annick Méjean ◽

Olivier Ploux

Keyword(s):

Molecular Oxygen ◽

Acyl Carrier Protein ◽

Three Dimensional ◽

Accessible Surface Area ◽

Polyketide Synthases ◽

Site Directed Mutagenesis ◽

Solvent Accessible Surface Area ◽

Dimensional Structure ◽

Structural Basis ◽

Carrier Protein

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 Å resolution. AnaB is a homotetramer and its fold is very similar to that of the acyl-CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl-CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl-CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes.

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