Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the

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Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase FabV Carla Maya Neckles, Annica Pschibul, Cheng-Tsung Eric Lai, Maria Hirschbeck, Jochen Kuper, Shabnam Davoodi, Junjie Zou, Nina Liu, Pan Pan, Sonam Shah, Fereidoon Daryaee, Gopal Reddy Bommineni, Cristina Lai, Carlos Simmerling, Caroline Kisker, and Peter J Tonge Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01301 • Publication Date (Web): 02 May 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase Carla Neckles1,2, Annica Pschibul6, Cheng-Tsung Lai3,4, Maria Hirschbeck6, Jochen Kuper6, Shabnam Davoodi1,2, Junjie Zou2,3, Nina Liu2, Pan Pan2, Sonam Shah2, Fereidoon Daryaee1,2, Gopal R. Bommineni2, Cristina Lai5, Carlos Simmerling1-4,*, Caroline Kisker6,*, and Peter J. Tonge1-2,4,* 1

Institute for Chemical Biology and Drug Discovery, 2Department of Chemistry, 3Laufer Center for Physical and Quantitative Biology and 4Graduate Program in Biochemistry and Structural Biology, Stony Brook University, Stony Brook, New York 11794, USA 5 6

William A. Shine Great Neck South High School, Great Neck, NY 11020, USA

Rudolf Virchow Center for Experimental Biomedicine, Institute for Structural Biology, University of Würzburg, D-97080 Würzburg, Germany

*Address correspondence to these authors: CS: Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794-5252 Tel: (631) 632 5424 Fax: (631) 632 5405 Email: [email protected] CK: Rudolf Virchow Center for Experimental Biomedicine, Institute for Structural Biology, University of Würzburg, D-97080 Würzburg, Germany Tel: +49 931 3180381 Email: [email protected] PJT: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400 Tel: (631) 632 7907 Fax: (631) 632 7934 Email: [email protected]

Keywords: enoyl-(acyl-carrier-protein) reductase, FabV, enzyme kinetics, binding kinetics, slowonset inhibition, induced-fit, molecular dynamics, X-ray crystallography

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ABSTRACT The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the present work, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20->100 fold). In addition, while T276S ypFabV generally displays higher affinity for 2-pyridone inhibitors compared to the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in gain of slow-onset inhibition for the 4-pyridone PT156.


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Two million people every year in the United States become infected with antibiotic resistant bacteria and about 23,000 Americans die per year because of these infections.1 The emergence of antimicrobial resistance is not only a concern in the United States but it is also seen as a serious global threat by the World Health Organization. The rise of antibiotic resistant bacteria poses even a greater health risk if these bacterial strains can be used in bioterrorism. One pathogen of interest is the Gram-negative bacterium Yersinia pestis because it is the causative agent of the plague and it has gained much attention due to its potential use as a biological warfare agent.2 This organism is now classified as a Tier 1 Biological Select Agent or Toxin (BSAT) by the Centers for Disease Control and Prevention (CDC). Although antibiotics such as streptomycin or doxycycline are effective in the treatment of Y. pestis infections, drug resistant strains of Y. pestis have been isolated that emphasize the need for novel chemotherapeutics.3 4-6 Fatty acids are essential components of bacterial cell membranes and enzymes in the type II fatty acid biosynthesis (FAS-II) pathway are promising targets for the discovery of novel therapeutics that are active against drug resistant strains.7 Although Brinster et. al. demonstrated that Gram-positive pathogens such as Streptococcus agalactiae can circumvent inhibition of the FAS-II pathway when supplied with exogenous fatty acids,8 Balemans et. al. found this does not hold for Staphylococcus aureus and confirmed the essentiality of the FAS-II pathway.9 Subsequently, Rock et al. demonstrated that the differential ability of S. agalactiae but not S. aureus to utilize fatty acid supplements resulted from suppression of de novo fatty acid synthesis in S. agalactiae via feedback inhibition of acetyl-CoA carboxylase.10 The importance of the FAS-II pathway for bacterial survival is also supported by the discovery of natural product inhibitors of fatty acid biosynthesis. For example, thiolactomycin and cerulenin target the β-


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ketoacyl-ACP synthases,11-13 while kalimantacin/batumin and pyridomycin inhibit the FabI enoyl-ACP reductase (ENR) isoform.14, 15 In addition, a significant number of synthetic FAS-II inhibitors have been reported, the majority of which target FabI.16 The front-line tuberculosis drug isoniazid inhibits the FabI ENR in Mycobacterium tuberculosis,17-19 whilst FabI inhibitors that are active in animal models of infection have been reported against pathogens including M. tuberculosis,20, 21 Staphylococcus aureus22, 23 and Francisella tularensis.24 Furthermore, AFN1252, an inhibitor of the S. aureus FabI, is currently in Phase II clinical trials.25 Thus, there is a strong support that the FabI ENR is a promising target for novel antibacterial discovery. In addition to FabI, three alternative ENR isoforms have been identified including FabK found in Streptococcus pneumoniae,26 FabL isolated from Bacillus subtilis27 and FabV identified in Vibrio cholerae.28 In contrast to the flavoprotein FabK—FabI, FabV and FabL are members of the short-chain dehydrogenase/reductase (SDR) superfamily and catalyze substrate reduction of the enoyl-ACP using NADH, or less commonly NADPH, as the hydride donor (Scheme 1). Most inhibitor discovery has focused on the FabI ENR, and the majority of FabI inhibitors require either the reduced or oxidized cofactor to be bound to the enzyme.16 Efforts to extend ENR inhibitor discovery to other pathogenic bacteria have been hindered by the presence of the alternative ENR isoforms that display differential sensitivity to current FabI inhibitors. FabV is less sensitive to the prototypical FabI ENR inhibitor triclosan, and the presence of both FabI and FabV isoforms in Pseudomonas aeruginosa is thought to be the reason for the reduced antibacterial activity of triclosan towards this organism.29 While some organisms have two ENR isoforms, Y. pestis only contains the FabV ENR (ypFabV). Given the success at developing antibacterial agents that act by inhibiting FabI, we previously performed an initial characterization of ypFabV with a focus on the T276S variant, a


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mutation adventitiously introduced during cloning.30 In this previous study, we demonstrated that triclosan was a poor inhibitor of T276S ypFabV with a Ki value of 71 µM. We also showed that the 2-pyridone inhibitors PT172 and PT173 had Ki values of 1-2 µM and we determined the X-ray structure of both compounds bound to FabV in the presence of NADH. In separate studies, a T276A mutant in Xanthomonas oryzae FabV (xoFabV) was found to have no detectable activity in enzyme assays.31 Interestingly, T276 is located at the N-terminus of the helical substrate-binding loop (T276-M284) in ypFabV, and this loop is known to be a key recognition element in the binding of substrates and inhibitors to the FabI ENRs.16 In the present work, we have performed a detailed characterization of ypFabV focusing on the function of T276 and its effect on substrate-binding loop dynamics to provide a foundation for structure-based inhibitor design.

We show that ypFabV catalyzes substrate

reduction via an ordered bi-bi mechanism with NADH binding first followed by the enoyl substrate. We also show that T276 plays a key role in the efficiency of substrate reduction by stabilizing the transition state for the reaction. Replacement of T276 with a serine alters the relative sensitivity of ypFabV for pyridone and diphenyl ether inhibitors, indicating that this residue modulates inhibitor recognition.

Subsequent structural analysis of enzyme-NADH

binary complexes in which T276 has been altered, provide insight into the dynamics of two loops (T276-M284 and R6-I11) that control access to the active site. Finally, we probe the molecular basis for time-dependent enzyme inhibition and demonstrate that replacement of P142 with a tryptophan residue results in gain of slow-onset inhibition for an inhibitor that displays rapidreversible binding kinetics for the wild-type enzyme. Molecular dynamics simulations and kinetic studies reveal that the P142W variant, which is located at the entrance to the major


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substrate binding portal, stabilizes the closed state of the active site, providing additional insight into the structural changes that drive drug-target residence time in the ENR enzyme family.


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METHODS Materials His-bind Ni2+-NTA resin was purchased from Invitrogen.

trans-2-Dodecenoic acid,

trans-2-decenoic acid, and trans-2-octenoic acid were purchased from TCI. Lauroyl-CoA was purchased from Sigma-Aldrich. Luria broth was obtained from VWR and Roth. Reagents for structural studies were purchased from Applichem, Carl Roth, Hampton Research and SigmaAldrich. All other chemical reagents were purchased from Fisher.

Synthesis of trans-2-dodecenoyl-CoA (ddCoA), trans-2-decenoyl-CoA (decCoA), and trans2-octenoyl-CoA (octCoA) The substrates ddCoA, decCoA, and octCoA were synthesized from trans-2-dodecenoic acid, trans-2-decenoic acid, and trans-2-octenoic acid, respectively, using the mixed anhydride method as previously described.18

The product was confirmed by negative ESI mass

spectrometry.

Compound synthesis Compounds PT01-05, PT10, PT12-13, PT15, PT70, PT91, PT113, PT155, PT156, PT157, PT166, PT171-173 and PT179 were available from previous studies.20, 32-34 Compounds PT424 and PT425 were synthesized using similar methods as the N-substituted 2-pyridones from former studies as described in supplementary information.34

Cloning, expression, and purification of ypFabV


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The fabV gene was amplified from the Y. pestis A1122 strain (BEI Resources NR-2644) using PCR with the primers listed in Table S1. The amplified PCR product was digested with XhoI and EcoRI and inserted into the pET15b vector (Novagen), such that a hexahistidine tag was encoded at the N-terminus. The sequence of the gene was confirmed by DNA sequencing (DNA Sequencing Facility, Health Science Center, Stony Brook University). Protein expression and purification were performed as described previously.30

The

concentration of FabV was determined spectrophotometrically at 280 nm using an extinction coefficient of 48,360 M-1 cm-1 calculated from the primary sequence of the protein (ExPASy ProtParam).

Site-directed mutagenesis, expression, and purification of ypFabV mutants The T276S variant was available from previous studies.30 The ypFabV mutants T276A, T276G, T276V, T276C, T276Y and P142W were prepared using the primers listed in Table S1. The amplified PCR product of the mutated fabV gene was digested with XhoI and EcoRI, and ligated into a pET15b vector. The sequence of each mutant plasmid was confirmed by DNA sequencing. All mutants were expressed in E. coli BL21(DE3) cells. A single colony resulting from transformation of E. coli with the relevant plasmid was used to inoculate 200 mL of LB containing 0.2 mg/mL ampicillin (LB/Amp). The culture was then incubated overnight at 37 °C (200 rpm) and subsequently used to inoculate 2 L of LB/Amp. Protein expression was then induced with 1 mM IPTG once the culture reached an OD600 of about 0.6. After incubating for 20 hr at 15 °C, cells were harvested by centrifugation and then frozen and stored at -20 °C. Prior to protein purification, the cells were thawed, re-suspended in 20 mM Tris-HCl pH 7.9 buffer


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containing 500 mM NaCl and 5 mM imidazole, and lysed. All subsequent chromatography steps were performed on the ÄKTA avant/pure system (GE Healthcare). The soluble fraction of the cell lysate obtained by centrifugation was loaded on to a HisTrap 5 mL FF crude affinity column (GE Healthcare) and eluted with 500 mM imidazole. Then, the hexahistidine tag was removed by overnight incubation at 4 °C with 1:100 (w/w) TEV protease in cleavage buffer (50 mM TrisHCl pH 7.5, 0.5 mM EDTA, 1 mM DTT). A second round of affinity chromatography (as described above) served to separate the cleaved product from the hexahistidine tag as well as any uncleaved protein.

The protein was further purified by size exclusion chromatography

(Superdex 200 26/60, GE Healthcare) using a buffer containing 20 mM PIPES pH 7.6, 300 mM KCl and 1 mM EDTA. The protein sample was concentrated, flash-frozen in liquid nitrogen and stored at −80°C.

Cloning, expression, and purification of E. coli ACP The acyl carrier protein (ACP) from E. coli (ecACP) was amplified by PCR using the primers listed in Table S1. The PCR amplified product was digested with EcoRI and XhoI, and ligated into the pET23b vector (Novagen) to encode a hexahistidine tag at the C-terminus of the protein. The construct was transformed, purified and sequenced as described above. Expression and purification of ecACP was performed as described previously using E. coli BL21(DE3) pLysS cells.35 The concentration of the protein was determined by measuring the absorption at 280 nm using an extinction coefficient of 1,490 M-1 cm-1. Conformationally sensitive gel electrophoresis on a non-denaturing 18% polyacrylamide gel containing 0.5 M Urea36 indicated that the ecACP was primarily in the apo form. LC-TOF mass spectrometry revealed that the apo-ecACP was comprised of two forms: apo ecACP in which the N-terminal Met was present


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(9462.57 Da) and in the second form in which the N-terminal Met had been cleaved (9331.37 Da).

Enzymatic preparation of trans-2-octenoyl-ecACP and trans-2-dodecenoyl-ecACP Apo-ecACP was converted to the acyl-ecACP using Sfp, the phosphopantetheinyl transferase from Bacillus subtilis, as previously reported.35 Briefly, 100 mg/mL apo-ecACP was incubated with 200 µM octCoA or ddCoA in the presence of 1 µM Sfp in 75 mM Tris-HCl pH 7.5 buffer containing 10 mM MgCl2, for 1 h at 37 °C. The reaction mixture was loaded onto a MonoQ 5/50 anion exchange column, and then eluted with a gradient of 100% buffer A (20mM Tris-HCl at pH 7.0) to 100% buffer B (20 mM Tris-HCl at pH 7.0 and 800 mM NaCl). Fractions were analyzed by conformationally sensitive gel electrophoresis,36 and those fractions that contained trans-2-octenoyl-ecACP (oct-ACP) or trans-2-dodecenoyl-ecACP (dd-ACP) were pooled and exchanged into 20 mM Tris-HCl, pH 7.5. The purified acylated protein was analyzed by LC-TOF or MALDI-TOF mass spectrometry.

MALDI-TOF: oct-ACP [M+23(Na)]+

9812.975 Da (acylated ACP without N-terminal Met). LC-TOF: dd-ACP 9983.25 Da (9852.25 Da without N-terminal Met).

Direct binding fluorescence titration experiments A fluorescence assay was used to determine the dissociation constant (Kd) of the substrate for wild-type and mutant forms of ypFabV. Titrations were performed at 25 °C and the change in fluorescence (excitation 280 nm) was monitored at 332 nm using a Quanta Master fluorometer (Photon Technology International). Excitation and emission slit widths were 4.0 and 2.0 mm, respectively. The substrate (stock concentration 1 mM ddCoA or 2.5 mM NADH) was titrated


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in 1 µL aliquots into a 0.5 µM enzyme solution (1 mL) prepared in pH 8.0 30 mM PIPES buffer containing, 150 mM NaCl and 1.0 mM EDTA. For T276Y and T276V ypFabV mutants, the stock concentration for NADH was increased to 25 mM to determine the Kd. The Kd values were determined by fitting the data to the Scatchard equation (Equation 1) using GraFit after the data were corrected for inner filter effects. In this equation, y is the amount of ligand bound per the amount of receptor; Cap is the capacity for the binding ligand; [L] is the concentration of the free ligand, and Kd is the dissociation constant of the ligand from the receptor.

Equation 1

Steady-state kinetic analysis Steady-state kinetics were performed on a Cary 100 Bio (Varian) spectrometer at 25 °C using 30 mM PIPES, 150 mM NaCl and 1.0 mM EDTA at pH 8.0 as the buffer.30 The reaction was followed by monitoring the oxidation of NADH to NAD+ at 340 nm (ε = 6,220 M-1 cm-1) and initial velocities were obtained as a function of one of the substrates whilst keeping the second substrate constant.

Characterization of the ypFabV mechanism was performed in

reaction mixtures containing 5 nM enzyme and obtaining initial velocities at fixed concentrations of NADH (50, 100, and 250 µM) and varying concentrations of ddCoA (1.5 - 24 µM), or at fixed concentrations of ddCoA (6, 12, and 24 µM) and varying concentrations of NADH (5 - 200 µM). Product inhibition studies were conducted in a similar manner except at a fixed concentration of one of the products lauroyl-CoA (0, 8, and 16 µM) or NAD+ (0, 50, and 100 µM) at subsaturating concentrations for the second substrate (50 µM NADH or 30 µM ddCoA). Characterization of the substrate binding mechanism for the ypFabV mutants was performed in a


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similar manner, except the enzyme concentration and varied substrate concentrations were adjusted based on the affinity and activity of the substrates for each mutant. Kinetic parameters for ypFabV were then determined by globally fitting the data in the absence of products to the equation for the steady-state sequential bi bi mechanism (Equation 2). In this equation, v is the initial velocity, Vmax is the maximum velocity, [A] and [B] are the concentration of the two substrates, KA and KB are the Michaelis constants for A and B respectively, and KiA is the dissociation constant of A. Data analysis was performed using GraphPad Prism.

Equation 2 Kinetic parameters were also obtained using a single-substrate kinetic study, in which one substrate was varied whilst maintaining the second substrate at a fixed saturating concentration. Datasets were fit to the Michaelis-Menten equation using Kaleidograph. Since the kinetic parameters for ypFabV were found to be very close to those obtained from global fitting, all subsequent kinetic parameters were determined using a single-substrate kinetic study. Inhibition constants were determined by non-linear regression analysis for competitive, uncompetitive, and noncompetitive inhibition using Equations 3-5, respectively, where [S] is the substrate concentration, [I] is the concentration of inhibitor added, KM is the Michaelis-Menten constant for the substrate, Vmax is the maximum velocity, and Ki and Ki' are the inhibition constants. The preferred inhibition model was determined based on statistical analysis of kinetic data including R2, standard errors, sum-of-squares and 95% confidence intervals.37

The

mechanism of inhibition for each inhibitor was additionally confirmed by Lineweaver-Burk plots.


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(Equation 3)

(Equation 4)

(Equation 5)

Inhibition kinetics IC50 values for the inhibition of wild-type and mutant forms of ypFabV were performed by adding varying concentrations of inhibitor dissolved in DMSO to reaction mixtures containing 250 µM NADH, 30 µM ddCoA and 15 nM enzyme. The total amount of DMSO added was kept constant (2% DMSO final). Initial velocities were obtained as a function of inhibitor concentration ([I]) and IC50 values were calculated by fitting the data to Equation 6 where y represents the percent activity.

(Equation 6) Progress curves for inhibition of P142W ypFabV by PT156 were determined in reaction mixtures that contained glycerol (8%), bovine serum albumin (0.1 mg/mL), DMSO (2% v/v), crotonyl-CoA (3.0 mM), NADH (0.25 mM), NAD+ (0.20 mM) and inhibitor (0-10 µM). The reactions were initiated by the addition of enzyme (10 nM) and the progress of the reaction was


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followed until the steady-state was reached, which was indicated by linearity of the progress curve.38-40 Progress curve data were fit to the integrated rate equation (Equation 7), where At and A0 are the absorbance at time t and 0; vi and vs are the initial and steady-state velocities; and kobs is the observed pseudo-first rate order constant for the approach to steady-state.24, 41

(Equation 7) Time-dependent inhibition was analyzed by assuming either a one or two step binding mechanism (Scheme 2). In the one-step mechanism, the forward and reverse rate constants for enzyme inhibition are k3 and k4, respectively.

For the two-step induced-fit slow binding

mechanism, rapid formation of the initial EI complex, described by Kiapp, is followed by a second slow isomerization leading to the final EI* complex, described by Ki*app and where the ratelimiting recovery of enzyme activity is assumed to be given by k6 (tR = 1/k6). In each case, the inhibition constants were determined by globally fitting the plots of fractional initial and steadystate velocities as a function of inhibitor concentration to Equation 7.

For a one-step

mechanism, vi = vo, and vs and kobs are defined by Equations 8-9. For a two-step mechanism, vi, vs and kobs are defined by Equations 10-12.

(Equation 8) (Equation 9)

(Equation 10) 14 ACS Paragon Plus Environment

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(Equation 11)

(Equation 12)

Crystallization and data collection Crystallization of wild-type ypFabV was performed as previously described.30 For the ypFabV T276A, T276G, T276C and T276V mutants, the protein was diluted to 60 mg/mL and preincubated with the cofactor NADH (ten-fold molar excess) for at least 1 hr. 1 +1 µL drops were equilibrated in hanging drop vapor diffusion experiments at 4 °C against 1 mL of the reservoir solution, containing 150 mM (NH4)2SO4, 100 mM MES pH 5.6-5.9 and 26.5-37.5 % PEG4000. Crystals were harvested after about two weeks and frozen in a cryo-protectant solution similar to the reservoir solution, but containing 10% DMSO or glycerol. All datasets were collected at a wavelength of 0.918 Å at beamlines MX 14.1 (Bessy, Berlin) and BM30A and ID 29 (ESRF, Grenoble).42

Structure determination and refinement Datasets were indexed and integrated with xds43 or imosflm44 and scaled in Scala45, 46 or aimless.47 All FabV structures were solved by molecular replacement with the program Phaser48 using the ypFabV (T276S) structure (PDB entry 3ZU3)30 as search model. The initial model was subsequently revised and adapted in Coot.49 Models were refined in Refmac50 and Phenix.51 TLS parameters used in refinement were created using the TLSMD server,52 and a library file supplying restraints for the NADH cofactor was created with the Prodrg server.53 The structures were validated using Phenix51 or the MolProbity server.54 15 ACS Paragon Plus Environment

Images were created using the

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program Pymol55 or Swiss-PDBViewer.56 Data collection and refinement statistics are given in (Tables S2 and S3).

Molecular dynamic simulation setup The wild-type (PDB entry 4BKR) and T276S ypFabV (PDB entry 3ZU5) were selected for molecular dynamic (MD) simulation studies.

30

structures

The P142W mutation was

introduced using the Swiss-PDBViewer.56 Ff99SB force fields were assigned to the protein,57 while the force field for the cofactor was taken from previous studies.58, 59 Each system was solvated in a truncated octahedral TIP3P water box with 8 Å distance between the solute and the water box edge.60 Bonds containing hydrogen were constrained using the SHAKE algorithm.61 The particle mesh Ewald method62 was used for calculating electrostatic energy with an 8 Å cutoff. All MD simulations were performed using GPU implemented AMBER12.63 All systems were equilibrated using the same procedure described in a previous study,64 except 10 additional dihedral angle restraints were applied to the inhibitor for the T276/S276 simulations (Table S4). The restraints employed for the T276/S276 simulations were based on a parabolic square-well potential65 that was comprised of (1) a well with a square bottom set to 30 deg; (2) two parabolic sides set to 60 deg with defined distances between (i) r1 and r2 with a force constant of k(R-r2)2 where k=10 kcal/deg2 [left parabola] and (ii) r3 and r4 with a force constant of k(R-r3)2 where k=10 kcal/deg2 [right parabola]; and (3) two linear sides beyond the parabolic sides such that one slope equals the derivative of the left parabola at r1 and the other slope equals the derivative of right parabola at r4. Production runs were performed for 300 ns for the simulations of the wild-type and P142W proteins, whereas 120 ns production runs were performed for the simulations of the T276 and S276 proteins using the above dihedral restraints.


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RESULTS Steady-state kinetics and mechanism of ypFabV Steady-state kinetic parameters were determined for ypFabV using both enoyl-CoA and enoyl-ACP substrates (Table 1). The value of kcat/KM for octenoyl-ACP is 75-fold larger than the value for octenoyl-CoA, indicating a preference for ACP over CoA-based substrates. In addition, as the chain-length of the enoyl-CoA substrate increased from C8 to C12, the kcat/KM increased from 2 to 390 µM-1min-1 (Table 1). These results reveal that ypFabV has a preference for longer chain enoyl substrates, in general agreement with the chain length specificity observed for the FabI ENRs.18, 35, 66 Next, we investigated the kinetic mechanism of ypFabV using the C12 substrate dodecenoyl-CoA (ddCoA) by measuring initial velocities at varying concentrations of ddCoA and NADH. Double-reciprocal plots show intersection of lines with each other, indicating a ternary complex mechanism (Figure 1 A, B). Furthermore, product inhibition studies with lauroyl-CoA and NAD+ were used to analyze the order of substrate binding and differentiate between ordered and random bi-bi mechanisms. At sub-saturating substrate concentrations, NAD+ is competitive with respect to NADH and noncompetitive with respect to ddCoA (Figure 1 C, D) while lauroyl-CoA is noncompetitive with respect to NADH and ddCoA (Figure 1 E, F). This product inhibition pattern is consistent with an ordered bi-bi mechanism in which NADH binds first to the enzyme followed by the enoyl substrate, consistent with other ENRs.18, 24, 67-69

The ternary complex steady-state kinetic parameters for ypFabV are Ki, NADH = 22 ± 3

µM, KM, NADH = 10 ± 1 µM, KM, ddCoA = 19 ± 1 µM and kcat = 5860 ± 388 min−1. These kinetic parameters, obtained from global fitting to Equation 2, are similar to the kinetic parameters obtained by varying one substrate at a fixed, saturating concentration of the second substrate


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(Table 2), and thus we subsequently determined kinetic parameters for the ypFabV mutants using single-substrate kinetic studies.

Significance of T276 in catalysis and stabilization of the transition state T276, located at the N-terminus of the substrate-binding loop, was found to be important for FabV catalysis in Xanthomonas oryzae.31 To explore the function of T276, the steady-state kinetic parameters following replacement of T276 with Ser, Gly, Ala, Val, Tyr and Cys mutants were compared to those for the wild-type enzyme (Table 2). The kcat/KM value of the T276S mutant is close to that of the wild-type enzyme. Replacement of T276 with Gly resulted in a 40fold reduction in kcat/KM, while the T276A, T276V and T276Y variants have kcat/KM values that are 390 to 1250-fold lower. The T276C ypFabV mutant had no detectable activity up to 850 nM enzyme and 240 µM ddCoA. However, direct binding experiments demonstrated that NADH (Kd = 1 µM) and ddCoA (Kd = 4 µM) could still bind to T276C ypFabV, in which the Kd values decreased by a factor of 13 and 3, respectively, compared to wild-type ypFabV. The activity of T276C ypFabV was then assayed with trans-2-dodecenoyl-ACP (dd-ACP), which has higher intrinsic affinity for the ENR enzymes than CoA-based substrates, and it was found that the T276C mutation reduced kcat/KM by 80,000-fold relative to reduction of the same substrate by wild-type ypFabV (819 to 0.01 µM-1min-1). Product inhibition studies for the T276 mutants were consistent with an ordered bi-bi mechanism similar to the wild-type enzyme (Figure S1-S5). Interestingly, the reduction in catalytic efficiency for all mutants, except T276S, was predominantly due to decreases in kcat, suggesting that the hydroxyl side-chain of T276 is important for stabilizing the transition state for substrate reduction.


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Biochemistry

Loop conformations upon ligand binding We solved the structures for the enzyme-NADH (E:NADH) binary complex for wildtype ypFabV, and the T276A, T276C, T276G and T276V ypFabV mutants (Tables S2 and S3). Although NADH binds in the same orientation in all E-NADH binary complexes, subtle differences were observed in specific interactions between NADH and the mutant enzymes (Figure 2A-F). The active site of ypFabV contains three catalytic residues, Y225, Y235 and K244 (Figure 2G-L). Mechanistic studies on the FabV from Burkholderia mallei suggested that Y235 interacts with the thioester carbonyl oxygen and has a hydrogen bond with K244, an interaction that would reduce the pKa of Y235 to facilitate the stabilization and protonation of the enolate intermediate formed during substrate reduction.69 However, in the FabV wild-type structure we observe that K244 exclusively interacts with the nicotinamide ribose moiety of the cofactor and Y235 interacts with the nicotinamide ribose as well via a water mediated hydrogen bond. The orientations of residues Y235 and K244 differ in three out of the five mutant structures, T276A, T276C and T276V. The side chain of Y235 rotates away from its original position so that the water-mediated cofactor interaction is no longer possible. In the three deviating variants K244 also assumes a different conformation leading to less well defined electron density and most likely to a loss of the interaction with the ribose. The change in orientation of Y235 may explain the dramatically reduced activity of the T276A, T276C and T276V mutants since this residue is thought to be important for stabilizing the transition state for substrate reduction.69 Superposition of all E-NADH binary complexes with reported ternary inhibitor FabV complexes (PDB entries 3ZU4 and 3ZU5) revealed two distinct conformations of the substratebinding loop (T276-M284) for the FabV enzyme (Figure 3). The E-NADH complexes for wild-


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type and T276S ypFabV are characterized by a closed active site conformation. In contrast, the substrate-binding loop changes its conformation in the T276A, T276C, T276G and T276V mutants, assuming an open state which resembles the inhibitor bound structure of the T276S mutant (Figure 3). In addition to changes in the substrate-binding loop, a second loop (R6-I11) located close to the substrate-binding loop, also alters its position when the active site widens. The R6-I11 loop contains a basic patch (K4, R6 and R8) and three residues within the hydrophobic patch (G9, F10, I11, V13, A15) that we previously suggested forms part of the ACP binding site.30 Analysis of the structures reveals that residues G9 and F10 are shifted away from the active-site depending on the conformation of loop R6-I11. These structural insights reveal that the substrate-binding loop and residues G9 and F10 may jointly contribute to FabV loop dynamics upon binding of the enoyl substrate and substrate mimic inhibitors.

Inhibitor structure-activity relationship (SAR) studies for ypFabV and T276S ypFabV Three related scaffolds have previously yielded inhibitors of the FabI ENR: the diphenyl ethers,32,

70, 71

2-pyridones72 and 4-pyridones.73-75 In the course of our own ENR inhibitor

discovery program, we synthesized analogues of each scaffold class and initially selected PT70 (a diphenyl ether),33 PT171 (a 2-pyridone)34 and PT166 (a 4-pyridone)34 for ypFabV inhibition studies. Each compound is structurally related and has a hexyl substituent on the A-ring and an o-CH3 on the B-ring (Table 3). PT70 is the most potent inhibitor of ypFabV (IC50 = 3 µM) compared to PT171 (IC50 > 50 µM) and PT166 (IC50 = 18 µM). In addition, PT70, PT171 and PT166 inhibit T276S ypFabV with IC50 values of > 100 µM, 6 µM and 23 µM, respectively. To expand our SAR studies we selected additional diphenyl ethers that vary in the nature of the A and B-ring substituents (Table 3). Comparison of inhibitors that differ based on the


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Biochemistry

alkyl substituent on the A-ring reveals that the propyl substituent (PT02) is the optimal size for ypFabV inhibition (IC50 = 0.2 µM), whereas compounds with shorter (e.g. PT01 IC50 = 20 µM) or longer (PT03-PT05 IC50 = 1 - 2 µM) alkyl substituents had reduced potency. Comparison of diphenyl ether inhibitors with an A-ring hexyl substituent revealed that introduction of an o-F (PT113, IC50 = 0.1 µM) or p-NO2 (PT12, IC50 = 0.2 µM) group into the B-ring resulted in significant improvements in potency. Finally, while most diphenyl ethers had no measurable activity against T276S ypFabV (IC50 > 100 µM), the introduction of substituents into the B-ring of the hexyl diphenyl ether scaffold resulted in some compounds that were able to inhibit this mutant (PT113 and PT12, IC50 5 - 8 µM). However these inhibitors remained significantly more potent for the wild-type enzyme, indicating that the methyl side-chain of T276 enhances diphenyl ether binding. In contrast to the diphenyl ethers, all 2-pyridones were better inhibitors of T276S ypFabV compared to wild-type ypFabV by at least a factor of 5, indicating that the methyl side-chain of T276 weakens binding of the 2-pyridones. Within this compound series, the addition of substituents at the ortho- or para-positions on the B-ring (PT179, PT171, PT172, PT424 and PT425) resulted in little improvement in the inhibition of ypFabV (IC50 = 21 - 56 µM) or T276S ypFabV (IC50 = 2 - 6 µM). PT173 (m-NH2, o-CH3 B-ring) was the exception, having no activity towards wild-type ypFabV (IC50 > 100 µM) but inhibiting T276S ypFabV with an IC50 value of 1 µM. In the 4-pyridone series, we observed that PT166 (B-ring o-CH3) has similar affinity for both wild-type and T276S ypFabV (IC50 ~20 µM). We also examined three additional 4pyridones (PT155, PT156 and PT157) and found that substitution of the B-ring with a p-NH2 and o-CH3 (PT155) decreased potency for ypFabV (IC50 > 50 µM), but increased potency for


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T276S ypFabV (IC50 = 1 µM). Upon the removal of the o-CH3 on the B-ring (PT157), inhibition of ypFabV improved by 250-fold (IC50 = 0.1 µM) and inhibition of T276S ypFabV improved 3fold (IC50 = 0.3 µM). Finally, the substitution of a p-NH2 with p-NO2 on the B-ring (PT156) did not have a significant effect on the IC50 for the wild-type enzyme, while there was a slight decrease in inhibitor potency for T276S ypFabV.

Mode of inhibition and cofactor preference To further explore the role of residue 276 in modulating the mechanism of enzyme inhibition, we selected structurally related inhibitors from each compound class that had reasonable IC50 values for both wild-type and T276S ypFabV, and performed a detailed kinetic analysis as summarized in Table 4. The representative compounds included the diphenyl ether PT12, the 2-pyridone PT424 and the 4-pyridone PT156 which each contained a p-NO2 B-ring substituent. The mode of inhibition for each compound was evaluated by analyzing the best fit of the experimental data to the equations for competitive, uncompetitive and noncompetitive inhibition (Table S5), and using Lineweaver-Burk plots (Figure S6). We found that the three compounds displayed different inhibition mechanisms for wild-type ypFabV: uncompetitive (PT12), noncompetitive (PT424) and competitive (PT156) with respect to ddCoA. In addition, PT156 and PT424 had the same mechanism of inhibition for T276S ypFabV, while noncompetitive inhibition was observed for PT12 (Table S5). The inhibition constants indicate that PT12 is a significantly better (~30-fold) inhibitor of wild-type FabV (Ki' = 0.1 µM) than of the T276S mutant (Ki' = 4 µM). In contrast, both PT424 and PT156 inhibit wild-type and T276S ypFabV with inhibition constants that differ by only ~3-fold (Table 4).


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Biochemistry

Diphenyl ethers and pyridones are known to bind to ENRs in the same location as the enoyl substrate.16 Thus, model fitting and Lineweaver-Burk plots show that the mechanism of inhibition is dependent on whether the inhibitors prefer to bind to the E-NADH or E-NAD+ binary complexes.

Scheme 3 summarizes the formation and breakdown of the inhibitor

complexes for wild-type and T276S ypFabV. PT156 is a competitive inhibitor of both wild-type and T276S ypFabV, consistent with the knowledge that the diphenyl ether class of ENR inhibitors bind preferentially to the E-NADH form of the enzyme.23,

67

In contrast, PT424

displays noncompetitive inhibition suggesting that the 2-pyridone inhibitor can bind to both the E-NADH and E-NAD+ binary cofactor complexes. Lastly, PT12 is an uncompetitive inhibitor of the wild-type enzyme but a noncompetitive inhibitor of T276S ypFabV with significantly reduced potency. Therefore, we hypothesize that the T276S mutant specifically destabilizes the interaction of the inhibitor with the binary cofactor product complex E-NAD+, such that binding to both E-NADH and E-NAD+ can be observed. The impact of the T276S mutant on diphenyl ether inhibition parallels that observed previously for resistance mutants of ecFabI.76, 77

Side-chain conformations for T276 and S276 To further explore the role of the T276 methyl side-chain in ligand binding, molecular dynamics (MD) simulations were performed on the E-NADH-PT173 ternary complexes with wild-type and T276S ypFabV. PT173 was chosen for these studies as we had previously determined the structure of this inhibitor bound to T276S ypFabV (PDB entry 3ZU5). Of the inhibitors studied in this work, PT173 has the highest affinity for T276S ypFabV (IC50 = 1 µM) but has no detectable inhibition of wild-type ypFabV (IC50 > 100 µM). Figure 4A shows a plot of residue 276 side-chain conformation for wild-type and T276S ypFabV over 120 ns of MD


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simulations.

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For the S276 side-chain, chi1 = 60° throughout the simulation, revealing a

preference of the gauche plus (g+) conformation. In contrast, chi1 starts at 60° and rotates to 180° for the T276 side-chain, revealing a preference for the trans (t) conformation. The Thr side-chain t rotamer is known to be unfavorable and normally observed 7% of the time.78 However, in this case the T276 side-chain conformation rotates from a g+ to t rotamer due to steric clashes between the methyl group of T276 and cofactor nicotinamide ring. This result coincides with direct binding experiments (Table 2), in which we observe a slight decrease in NADH affinity for wild-type ypFabV (Kd = 13 µM) compared to the T276S mutant (Kd = 4 µM). Superposition of wild-type and T276S ypFabV structures following MD simulations with the crystal structure of T276S ypFabV (PDB entry 3ZU5) yielded RMSD values of 1 and 1.33 Å, respectively (Figure S7).

The simulations reveal that alteration in the T276 rotamer

conformation causes the T276 methyl group to displace the structured water molecule that bridges the hydroxyl side-chain of T276 and the backbone carbonyl of S279 (Figure 4B). All water molecules were replaced with bulk water in the simulation, and thus this observation is not due to the restraints from the input structure. In addition, the methyl group of T276 clashes with the backbone carbonyl of S279 in the t rotamer, and this unfavorable interaction leads to an increase in the distance between S279 and the T276 methyl group from 2.5 Å to 4.5 Å (Figure S7B). Based on this result, we speculate that T276 disrupts the hydrogen bonding network between S276, S279 and structured water molecules that stabilizes interactions between NADH and the A-ring of the 2-pyridones PT172 and PT173 when these inhibitors are bound to T276S ypFabV.30 Disruption of this network may thus explain why many 2-pyridone inhibitors have reduced affinity for wild-type ypFabV compared to the T276S mutant.


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Biochemistry

Slow onset inhibition of P142W ypFabV Many slow-onset inhibitors have been discovered for the FabI ENR, however, none of the compounds described here are slow-onset inhibitors of ypFabV. Slow-onset inhibition is however observed for inhibitors of the ENR from M. tuberculosis (InhA), and previously we demonstrated that a change in conformation of the substrate-binding loop, from an open to a closed state, was responsible for time-dependent inhibition in this system.64 In ypFabV we also observe open and closed conformations of the substrate-binding loop, indicating that the loop in this enzyme is also able to adopt alternative positions depending on the liganded state of the enzyme. Changes between the open and closed states in ypFabV alter the size of the major portal, which defines the entrance into the active site, and we analyzed the available structural data to identify residues that might stabilize the closed state of the loop when inhibitors are bound. This analysis revealed that P142 may serve as a gatekeeper for ligand binding, and that replacing P142 with a residue that has a bulkier side-chain, such as tryptophan, might create a “door” to form a closed active site conformation when the inhibitor is bound to the enzyme (Figure 5A, B). The distance between the side-chain of residue 142 and the backbone atoms of residues 279, 281 and 282 was used as a quantitative metric to analyze alterations in the size of the major portal. This distance is ~13 Å in the open conformation observed in the structure of PT173 bound to ypFabV (PDB entry 3ZU5), and ~10 Å in the closed conformation observed in the ypFabV NADH binary complex (PDB entry 4BKR). To gain insight into the effect of enlarging the side-chain of P142 on the major portal, MD simulations were performed on the structure of wild-type ypFabV complexed with NADH (PDB entry 4BKR), and the same structure after replacing P142 with a tryptophan. In the wild-type enzyme the distance varies between 8.5 and 13 Å with a maximum probability at 9.9 Å (Figure 5C,F), whilst this distance


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drops to 5-10 Å for the P142W mutant in which states with distances of 6-8 Å have the most significant contributions (Figure 5D,E,G). Thus, the MD simulations suggest that the P142W should have a more occluded active site. We thus generated and characterized the P142W ypFabV mutant.

The steady-state

kinetic parameters for P142W ypFabV are similar to values for wild-type ypFabV (Table 2), and product inhibition studies revealed that lauroyl-CoA is noncompetitive with respect to NADH and ddCoA, as observed for the wild-type enzyme (Figure S8). Thus the P142W mutation has not significantly altered the catalytic efficiency of the enzyme. However, progress curves for the inhibition of P142W ypFabV by PT156 were consistent with slow-onset kinetics (Figure 6). Progress curves obtained at different inhibitor concentrations were globally fit to Equation 7 for one-step (Scheme 2A) and two-step (Scheme 2B) slow-binding mechanisms. The best-fit was obtained for a two-step mechanism, giving a residence time (tR) of 78 min for the PT156-P142W ypFabV complex (Table 5). In contrast, progress curves of wild-type ypFabV in the presence of PT156 were linear, consistent with rapid binding kinetics (data not shown). Analyses of the inhibition constants indicated that the Ki*app for inhibition of P142W by PT156 is larger than the Ki for inhibition of the wild-type enzyme (Tables 4 and 5). Thus, the overall thermodynamic stability of the enzyme-inhibitor complex is actually lower for P142W ypFabV and slow-onset inhibition is caused by destabilization of the transition state leading to the final EI* complex.

DISCUSSION We previously analyzed the inhibition of S276 ypFabV by two 2-pyridone inhibitors using kinetic and structural approaches. Although we cloned the gene for ypFabV from genomic DNA in which residue 276 was reported to be a threonine, as found in the sequence of the wild-


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Biochemistry

type enzyme, cloning apparently introduced a mutation changing T276 to a serine. This was interesting because Li et. al. had reported that the T276A mutation in the FabV from X. oryzae (xoFabV) dramatically reduced the activity of the enzyme.31

Consequently, we set out to

characterize wild-type ypFabV and to explore the role of residue 276 in substrate reduction and enzyme inhibition. FabV is a member of the SDR superfamily and typically substrate reduction for ENRs within this family occurs via a ternary complex mechanism.18, 24, 67-69

33, 34

In agreement with

previous studies, our data indicate that ypFabV catalyzes substrate reduction through an ordered bi-bi ternary complex mechanism in which NADH binds first to the enzyme (Figure 1). In addition, as expected, the catalytic efficiency of ypFabV is significantly higher for enoyl-ACPs compared to the corresponding CoA-based substrates (Table 1). However, for ypFabV the increase in kcat/KM for ACP-based substrates is driven by an increase in kcat, rather than a reduction in KM as is normally observed in the ENR family.18,

66, 79

Thus remote binding

interactions between ACP and ypFabV contribute to catalysis, as observed in other systems such as the classic example of 3-oxoacid coenzyme A transferase.80 Although wild-type and T276S ypFabV have similar catalytic efficiencies, most T276 mutants have significantly reduced activity compared to the wild-type enzyme. The effect of these mutations on the enzyme structure was evaluated by X-ray crystallography, which revealed that the substrate-binding loop adopts a closed conformation for binary E-NADH complexes of wild-type and T276S ypFabV, whereas this loop is in a more open conformation for the other T276 mutants studied here (Figure 3). Thus the mutations have altered the relative stability of conformations that can be adopted by the substrate-binding loop. In addition, in several mutant enzymes the position of the active site Tyr and Lys residues is also altered, and thus there is


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evidence of alterations to the active site which may contribute to the decrease in activity observed for these enzymes. SAR studies with three related inhibitor scaffolds demonstrate that T276 also has an impact on enzyme inhibition. Previous inhibition studies with the FabI isoform resulted in the general notion that the diphenyl ethers likely bind as the anion (phenolate) to the E-NAD+ product complex, and thus are transition state analogs of the enzyme catalyzed reaction.16, 66, 77 In contrast, the pyridones are substrate-like and bind to the E-NADH form of the enzyme.23, 30 The studies here show that diphenyl ethers have significantly higher affinity for the wild-type ypFabV than for the T276S mutant, while conversely, 2-pyridones are generally better inhibitors of T276S ypFabV (Table 3). MD simulations reveal that in the wild-type enzyme the T276 sidechain adopts a t conformation, which we speculate may facilitate a favorable hydrophobic interaction between M285, which is close to the substrate-binding loop, and the A-ring of the diphenyl ether. Direct hydrophobic interactions between the inhibitor and the substrate-binding loop are not possible if the T276 side-chain is in the g+ conformation. The 2-pyridones likely bind less potently to wild-type ypFabV since the T276 methyl group disrupts a hydrogenbonding network that stabilizes the T276S ypFabV-cofactor interactions (Figure 4).

In

agreement with previous studies in which we demonstrated that a 4-pyridone had broader activity against the FabI enzymes from different organisms compared to 2-pyridones,23 here we also find that in general binding of 4-pyridones to ypFabV is less affected by specific changes to the active site architecture such as the T276 to S mutation. The structural analysis of ypFabV inhibition led to a hypothesis that we could modulate access to the active site by altering residue P142.

Computational studies predicted that

replacement of P142 with a tryptophan residue would reduce the size of the major portal, and


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Biochemistry

experimentally we observed that this mutation destabilizes both the ground state of the enzymeinhibitor complex as well as the transition state on the inhibition reaction coordinate such that PT156 is now a time-dependent inhibitor of P142W ypFabV (Table 5 and Figure 5). By analogy to the InhA system, we hypothesize that transitions between the open and closed states of the substrate-binding loop in ypFabV control the kinetics of enzyme inhibition, and that the P142W mutation has introduced an energy barrier between these states such that time-dependent inhibition is now observed. The ability to engineer time-dependent inhibition into ypFabV provides a foundation for understanding the molecular factors that modulate the life time of enzyme-inhibitor complexes in this and other enzyme families.


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SUPPORTING INFORMATION Supporting information includes data collection and refinement statistics for the X-ray crystallographic studies, additional steady-state kinetic plots and synthetic methods.

This

material is available free of charge via the Internet at http://pubs.acs.org.

FUNDING SUPPORT This work was supported in part by the National Institutes of Health (grant GM102864 to P.J.T.) and by the Deutsche Forschungsgemeinschaft (grants SFB630 to C.K. and C.A.S. and Forschungszentrum FZ82 to C.K.). C.N. was supported by the Chemical-Biology Interface Training Program grant (NIH T32GM092714) and SUNY LSAMP Bridge to the Doctorate (BD) Cohort at Stony Brook (NSF HRD0929353).

ACKNOWLEDGMENT We thank the staff of BL 14.1 at BESSYII, Berlin and ID29 of the ESRF, Grenoble for technical support, along with Dr. Béla Ruzsicka from the Institute of Chemical Biology and Drug Discovery for mass spectroscopy instrumentation. In addition, we acknowledge the Stony Brook University Proteomics Center.

REFERENCES [1] (2013) Antibiotic Resistance Threats in the United States, Centers for Disease Control and Prevention.


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[2] Richards, J. L., and Grimes, D. E. (2008) Bioterrorism: Class A agents and their potential presentations in immunocompromised patients, Clin J Oncol Nurs 12, 295-302. [3] Butler, T. (2009) Plague into the 21st century, Clin Infect Dis 49, 736-742. [4] Galimand, M., Carniel, E., and Courvalin, P. (2006) Resistance of Yersinia pestis to antimicrobial agents, Antimicrob Agents Chemother 50, 3233-3236. [5] Guiyoule, A., Gerbaud, G., Buchrieser, C., Galimand, M., Rahalison, L., Chanteau, S., Courvalin, P., and Carniel, E. (2001) Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis, Emerg Infect Dis 7, 43-48. [6] Guiyoule, A., Rasoamanana, B., Buchrieser, C., Michel, P., Chanteau, S., and Carniel, E. (1997) Recent emergence of new variants of Yersinia pestis in Madagascar, J Clin Microbiol 35, 2826-2833. [7] Heath, R. J., and Rock, C. O. (2004) Fatty acid biosynthesis as a target for novel antibacterials, Curr Opin Investig Drugs 5, 146-153. [8] Brinster, S., Lamberet, G., Staels, B., Trieu-Cuot, P., Gruss, A., and Poyart, C. (2009) Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens, Nature 458, 83-86. [9] Balemans, W., Lounis, N., Gilissen, R., Guillemont, J., Simmen, K., Andries, K., and Koul, A. (2010) Essentiality of FASII pathway for Staphylococcus aureus, Nature 463, E3; discussion E4. [10] Parsons, J. B., Frank, M. W., Subramanian, C., Saenkham, P., and Rock, C. O. (2011) Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors, Proc Natl Acad Sci U S A 108, 15378-15383.


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Scheme 1. The reaction catalyzed by ypFabV.


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Biochemistry

Scheme 2. One and two-step inhibition mechanisms.


Biochemistry

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Scheme 3. Formation and breakdown of inhibitor complexes for wild-type (blue) and T276S (red) ypFabV. E=free enzyme; S=substrate; P=product. E-NAD +-PT12 E-NAD +-PT424

E-NADH-PT156 E-NADH-PT424

E+NADH

E-NADH+S

E-NAD +-P

E-NADH-S

E-NADH-PT12 E-NADH-PT156 E-NADH-PT424

E-NAD ++P

E-NAD +-PT12 E-NAD +-PT424


E+NAD ++P

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Biochemistry

Table 1. Kinetic parameters for the reduction of CoA and ACP-based substrates by ypFabV.

a

Substratea trans-2-octenoyl-ecACP

KM (µM) 6±2

kcat (min-1) 895 ± 125

kcat/KM (µM-1min-1) 150 ± 53

trans-2-octenoyl-CoA

7±1

16 ± 1

2±0

trans-2-decenoyl-CoA

23 ± 3

332 ± 20

14 ± 2

trans-2-dodecenoyl-CoA

14 ± 1

5468 ± 426

390 ± 41

ecACP: E. coli acyl-carrier-protein.


Biochemistry

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Table 2. Kinetic parameters for wild-type ypFabV and mutants. Kd (µM)b NADH ddCoA 13 ± 1 13 ± 1

5468 ± 426

kcat/KM (µM-1min-1)a 390 ± 41

33 ± 0

5034± 445

280 ± 40

11 ± 0

3±0

133 ± 9

1±0

38 ± 6

2±0

47 ± 4

359 ± 24

10 ± 2

T276C ypFabVc

—

1±0

4±0

—

—

T276V ypFabV

17 ± 3

112 ± 4

3±1

15 ± 1

1±0

T276Y ypFabV

15 ± 2

120 ± 1

2±0

5±0

0.3 ± 0

P142W ypFabV

12 ± 2

6±0

56 ± 2

3866 ± 243

349 ± 60

Enzyme

KM, ddCoA (µM)a

Wt ypFabV

14 ± 1

T276S ypFabV

18 ± 2

4±0

T276A ypFabV

53 ± 5

T276G ypFabV

a

kcat (min-1)a

Parameters determined by steady-state kinetic analysis. Parameters determined by direct binding fluorescence titration experiments. c Mutant activity was not detectable under assay conditions. b


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Biochemistry

Table 3. IC50 values for inhibition of wild-type and T276S ypFabV. Inhibitor

Structure

IC50 (µM)a Wt ypFabV T276S ypFabV Diphenyl Ethers

PT01

20 ± 3

>100

PT02

0.2 ± 0

>100

PT03

1±0

>100

PT04

3±0

>100

PT05

2±0

>100

PT10

1±0

68 ± 14

PT12

0.2 ± 0

5±1

PT13

2±0

45 ± 3

PT15

3±0

>100

PT70

3±1

> 100

PT91

4±1

>100

PT113

0.1 ± 0

8 ±2


Biochemistry

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Inhibitor

Structure

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IC50 (µM)a Wt ypFabV T276S ypFabV 2-Pyridones

PT171

56 ± 6

6±1

PT172

55 ± 6

2±0 Ki,c = 2 ± 0 30

PT173

>100

1±1 Ki,c = 2 ± 0 30

PT179

42 ± 9

3±0

PT424

21 ± 7

3±0

PT425

30 ± 5

6±1

4-Pyridones

a

PT155

>50

1±0

PT156

0.2 ± 0

1±0

PT157

0.1 ± 0

0.3 ± 0

PT166

18 ± 2

23 ± 2

IC50 determined using 15 nM enzyme, 250 µM NADH and 30 µM ddCoA.


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Biochemistry

Table 4. Mode of inhibition and binding constants for selected compounds.a

PT12 Enzyme

PT424

PT156 Inhibition constants (µM)

Mode of Inhibition

PT12

PT156

PT424

PT12

Wt ypFabV

Ki' = 0.1 ± 0

Ki = 0.2 ± 0

Ki = 8 ± 2 Ki' =16 ± 5

U

C

N

T276S ypFabV

Ki = 8 ± 4 Ki' = 4 ± 2

Ki = 0.5 ± 0

Ki = 3 ± 0 Ki' = 3 ± 0

N

C

N

a

PT156 PT424

Inhibition parameters and mechanism of inhibition for each compound with respect to ddCoA were determined by best fit of the data to equations 3-5 (Table S5) and by double reciprocal plots (Figure S6). N= noncompetitive; U=uncompetitive; C=competitive


Biochemistry

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Table 5. Kinetic parameters for inhibition of P142W ypFabV by PT156.a tR (min)b 78 ± 6

konapp (overall) (µM-1min-1)c 0.022 ± 0.002

Kiapp (µM)d 6±0

k6 (min-1)d 0.013 ± 0.001

Ki*app (µM)d 0.6 ± 0.01

a

Parameters were determined using 10 nM enzyme, 3 mM crotonyl-CoA, 0.25 mM NADH and 0.20 mM NAD+ at fixed inhibitor concentrations (0-10 µM). b Calculated using tR =1/k6. c Calculated using konapp = k6/Ki*app. d All datasets at fixed inhibitor concentrations were globally fit using Equation 7, in which vi, vs and kobs were defined by Equations 10-12.


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Biochemistry

Figure Legends:

Figure 1. Two-substrate steady-state kinetics and product inhibition studies to determine the substrate binding mechanism of wild-type ypFabV. Initial velocity patterns: (A) 1/v versus 1/[ddCoA] in which [NADH] was fixed at 50 (○), 100 () and 250 ( ) µM and (B) 1/v versus 1/[NADH] in which [ddCoA] was fixed at 6 (○), 12 () and 24 ( ) µM. Product inhibition studies were performed at 50 µM NADH or 30 µM ddCoA at varying concentrations of the second substrate.

Initial velocity patterns: (C) 1/v versus

1/[ddCoA] and (D) 1/v versus 1/[NADH] at 0 (○), 50 (), and 100 ( ) µM NAD+. (E) 1/v versus 1/[ddCoA] and (F) 1/v versus 1/[NADH] at 0 (○), 8 () and 16 ( ) µM lauroyl CoA.

Figure 2. The active site of the ypFabV T276 mutants shows different conformations of important residues and a loss of interactions with the cofactor compared to the wild-type. (A-F) Interactions of residue 276 and its neighbors. In the wild-type (A, brown) and the T276S mutant (B, wheat), the hydroxyl group of residue 276 interacts via hydrogen bonding with the pyrophosphate of NADH. This interaction is absent in T276A (C, blue), T276C (D, firebrick), T276G (E, deep teal) and T276V (F, violet purple). (G-L) Orientation of the catalytically important residues Y225, Y235 and K244. The color scheme is similar to the upper panel. In the structures of wild-type (G) and T276S (H) ypFabV, Y235 and K244 interact with the nicotinamide moiety of the cofactor, in the case of Y235 via an ordered water molecule. These interactions can also be found in the T276G structure (K), but a MES molecule from the crystallization buffer takes the place of the ordered water molecule. In some structures, other molecules from the crystallization buffer are also present (e.g. DMSO), but these do not


Biochemistry

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contribute to cofactor binding. In the structures of the other mutants, residues Y235 and K244 show orientations which are not beneficial for interaction with the cofactor.

Figure 3: The ypFabV T276A, T276C, T276G and T276V mutants mimic the substratebinding loop conformation found in the ternary inhibitor complexes. In inhibitor-bound ypFabV complex (PDB code 3ZU4, green), the substrate-binding loop and an adjacent loop near the N-terminus of ypFabV move away from the active site, forming an open state compared to the wild-type (brown) and T276S (wheat) structures. The binary NADH complexes of the T276A (blue), T276C (firebrick), T276G (deep teal) and T276V (violet purple) mutants also exhibit this open active site, even though no inhibitor is bound. The substratebinding loop adopts a closed conformation in the structures of wild-type and T276S ypFabV bound to NADH. The loops in question are shown in ribbon representation.

Figure 4. Comparison of the T276S ypFabV-NADH-PT173 X-ray structure with that obtained following MD simulations of the wild-type enzyme and Ser mutant. MD simulations of the ternary inhibitor complex of E-NADH-PT173 were performed for T276 and T276S ypFabV over 120 ns. (A) A plot of chi1 for residue 276 over 250 MD frames reveal that the S276 side-chain rotamer remains in the g+ conformation throughout the MD simulation, while the T276 side-chain conformation rotates from a g+ to t rotamer after 50 frames. (B) A representative frame from the MD simulations (frame 89) was selected for comparative analysis after T276 side-chain rotates to the t conformation and ypFabV structures generated by MD simulations for the wild-type enzyme (blue) and T276S mutant (cyan) bound to NADH and 2pyridone PT173 was aligned with the crystal structure of the ternary inhibitor T276S ypFabV


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Biochemistry

complex (PDB entry 3ZU5, wheat). The structured water molecule bridging the hydroxyl sidechain of T276 and the backbone carbonyl of S279 is displaced by the introduction of the methyl group. Hydrogen bonding interactions are represented by dashes and all water molecules are depicted as spheres.

Figure 5. The major portal for wild-type and P142W ypFabV. (A) The x-ray structure of NADH bound to ypFabV (PDB entry 4BKR) reveals the location of P142 (yellow space filling) at the entrance of the active site pocket. P142 thus serves as a putative gate keeper for the major portal. (B) Residue P142 has been replaced with a tryptophan (yellow space filling) to show how a bulky side-chain could cover the substrate entrance to stabilize the closed state of the substrate-binding loop when inhibitors are bound. (C-E) MD simulations of the binary E-NADH complexes for both wild-type ypFabV (C) and the P142W mutant (D and E) revealed variation in distance across the entrance of the major portal. (F and G) The distance between the side-chain of residue 142 (yellow) and backbone of residues 279, 281, and 282 (blue) was used to map the probability for alterations in the size of the major portal throughout the MD simulations.

Figure 6. Determination of the inhibitor mechanism of the 4-Pyridone PT156 for P142W ypFabV. Progress curves for P142W ypFabV in the presence of 0-10 µM PT156 where datasets were globally fitted to Equation 7 for a two-step slow-binding mechanism (R2 = 0.99).


Biochemistry

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Figure 1. Two-substrate steady-state kinetics and product inhibition studies to determine the substrate binding mechanism of wild-type ypFabV.


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Biochemistry

Figure 2. The active site of the ypFabV T276 mutants shows different conformations of important residues and a loss of interactions with the cofactor compared to the wild-type.

55

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Figure 3.

The ypFabV T276A, T276C, T276G and T276V mutants mimic the substrate-

binding loop conformation found in the ternary inhibitor complexes.


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Biochemistry

Figure 4. Comparison of the T276S ypFabV-NADH-PT173 X-ray structure with that obtained following MD simulations of the wild-type enzyme and Ser mutant.


Biochemistry

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Figure 5. The major portal for wild-type and P142W ypFabV.


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Biochemistry

Figure 6. Determination of the inhibitor mechanism of the 4-Pyridone PT156 for P142W ypFabV.


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FOR TABLE OF CONTENTS USE ONLY Manuscript title: Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for Yersinia pestis FabV Enoyl-ACP Reductase Authors: Carla Neckles, Annica Pschibul, Cheng-Tsung Lai, Maria Hirschbeck, Jochen Kuper, Shabnam Davoodi, Junjie Zou, Nina Liu, Pan Pan, Sonam Shah, Fereidoon Daryaee, Gopal R. Bommineni, Cristina Lai, Carlos Simmerling, Caroline Kisker, and Peter J. Tonge

E-NADH-Inhibitor Mimic Slow Binding

E-NADH

β9/β10

E-NADH-Inhibitor Mimic Rapid Reversible

β7/β8 SBL NADH β1/β2

P142W ypFabV

Wild-type ypFabV T276S ypFabV


T276A/G/V/C ypFabV

Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the

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