Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309874200
ISSN1083-351X
AutoresYongmei Zhang, Bainan Wu, Jie Zheng, Charles O. Rock,
Tópico(s)RNA and protein synthesis mechanisms
ResumoFatty acid synthesis in bacteria is catalyzed by a set of individual enzymes collectively known as type II fatty-acid synthase. Each enzyme interacts with acyl carrier protein (ACP), which shuttles the pathway intermediates between the proteins. The type II enzymes do not possess primary sequence similarity that defines a common ACP-binding site, but rather are hypothesized to possess an electropositive/hydrophobic surface feature that interacts with the electronegative/hydrophobic residues along helix α2 of ACP (Zhang, Y.-M., Marrakchi, H., White, S. W., and Rock, C. O. (2003) J. Lipid Res. 44, 1-10). We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on β-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix α2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix α2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably. Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes collectively known as type II fatty-acid synthase. Each enzyme interacts with acyl carrier protein (ACP), which shuttles the pathway intermediates between the proteins. The type II enzymes do not possess primary sequence similarity that defines a common ACP-binding site, but rather are hypothesized to possess an electropositive/hydrophobic surface feature that interacts with the electronegative/hydrophobic residues along helix α2 of ACP (Zhang, Y.-M., Marrakchi, H., White, S. W., and Rock, C. O. (2003) J. Lipid Res. 44, 1-10). We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on β-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix α2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix α2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably. Fatty acid biosynthesis in bacteria and plants is carried out by a series of enzymes that are collectively known as the type II fatty-acid synthase and is most extensively studied in the Escherichia coli system (1Heath R.J. Jackowski S. Rock C.O. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins, and Membranes. 4th Ed. Elsevier Science Publishing Co., Inc., New York2002: 55-92Google Scholar, 2Jackowski S. Rock C.O. Biochem. Biophys. Res. Commun. 2002; 292: 1155-1166Crossref PubMed Scopus (162) Google Scholar). ACP 1The abbreviations used are: ACPacyl carrier proteinFabfatty acid biosynthesis enzymeFabHβ-ketoacyl-ACP synthase IIIFabGβ-ketoacyl-ACP reductaseFabDmalonyl-CoA:ACP transacylaseAcpSholo[ACP]synthase.1The abbreviations used are: ACPacyl carrier proteinFabfatty acid biosynthesis enzymeFabHβ-ketoacyl-ACP synthase IIIFabGβ-ketoacyl-ACP reductaseFabDmalonyl-CoA:ACP transacylaseAcpSholo[ACP]synthase. is a small, acidic protein that functions as the central acyl group carrier in type II fatty-acid synthase systems. The ACPs are rod-shaped proteins with a preponderance of acidic residues organized into four α-helices (3Holack T.A. Nilges N. Prestegard J.H. Gronenborn A.M. Clore G.M. Eur. J. Biochem. 1988; 179: 9-15Crossref Scopus (72) Google Scholar, 4Kim Y.M. Prestegard J.H. Protein Struct. Funct. Genet. 1991; 8: 377-385Crossref Scopus (156) Google Scholar, 5Kim Y. Ohlrogge J.B. Prestegard J.H. Biochem. Pharmacol. 1990; 40: 7-13Crossref PubMed Scopus (17) Google Scholar, 6Crump M.P. Crosby J. Dempsey C.E. Parkinson J.A. Murray M. Hopwood D.A. Simpson T.J. Biochemistry. 1997; 36: 6000-6008Crossref PubMed Scopus (141) Google Scholar, 7Wong H.C. Liu G. Zhang Y.M. Rock C.O. Zheng J. J. Biol. Chem. 2002; 277: 15874-15880Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The acyl intermediates are attached to the terminal sulfhydryl of the 4′-phosphopantetheine prosthetic group (8Prescott D.J. Vagelos P.R. Adv. Enzymol. Relat. Areas Mol. Biol. 1972; 36: 269-311PubMed Google Scholar), which is attached via a phosphodiester linkage to Ser-36 located at the beginning of the second helical segment. The primary gene product is an apoprotein that is converted to ACP by the transfer of the 4′-phosphopantetheine moiety of CoA to Ser-36 by [ACP]synthase (AcpS) (9Flugel R.S. Hwangbo Y. Lambalot R.H. Cronan Jr., J.E. Walsh C.T. J. Biol. Chem. 2000; 275: 959-968Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 10Parris K.D. Lin L. Tam A. Mathew R. Hixon J. Stahl M. Fritz C.C. Seehra J. Somers W.S. Structure. 2000; 8: 883-895Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). ACP performs two functions. First, it sequesters the growing acyl chain from the aqueous environment, and second, upon binding to one of the type II proteins, it releases its grip on the fatty acid, which is inserted into the active site cavity of the enzyme. ACPs are widely distributed in nature and are easily recognized by their significant primary sequence identity, particularly at the prosthetic group attachment site and extending along helix α2 (11Zhang Y.-M. Marrakchi H. White S.W. Rock C.O. J. Lipid Res. 2003; 44: 1-10Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These similarities are thought to underlie the observation that ACPs from virtually any source are substrates for AcpS and function with the fatty acid biosynthetic enzymes of E. coli (11Zhang Y.-M. Marrakchi H. White S.W. Rock C.O. J. Lipid Res. 2003; 44: 1-10Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, the individual enzymes of type II fatty acid synthesis do not share a primary structure motif that would define the presence of a common ACP-binding motif. acyl carrier protein fatty acid biosynthesis enzyme β-ketoacyl-ACP synthase III β-ketoacyl-ACP reductase malonyl-CoA:ACP transacylase holo[ACP]synthase. acyl carrier protein fatty acid biosynthesis enzyme β-ketoacyl-ACP synthase III β-ketoacyl-ACP reductase malonyl-CoA:ACP transacylase holo[ACP]synthase. The molecular details that govern the specific interactions between ACP and the type II enzymes are poorly known. The analysis of the binding of ACP to FabH points to ionic interactions playing a determinant role in the interaction between these two proteins (12Zhang Y.-M. Rao M.S. Heath R.J. Price A.C. Olson A.J. Rock C.O. White S.W. J. Biol. Chem. 2001; 276: 8231-8238Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This study used a computational algorithm to dock the NMR structure of E. coli ACP to FabH. ACP was predicted to dock to a hydrophobic patch with embedded positively charged residues adjacent to the active site tunnel. Kinetic analysis and direct binding studies between FabH mutants and ACP confirmed the importance of the positively charged residues to the FabH-ACP interaction. Several ACP mutants have been made, and their ability to function in type II fatty acid synthesis has been assessed (13Flaman A.S. Chen J.M. Van Iderstine S.C. Byers D.M. J. Biol. Chem. 2001; 276: 35934-35939Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Worsham L.M. Earls L. Jolly C. Langston K.G. Trent M.S. Ernst-Fonberg M.L. Biochemistry. 2003; 42: 167-176Crossref PubMed Scopus (40) Google Scholar, 15Gong H. Byers D.M. Biochem. Biophys. Res. Commun. 2003; 302: 35-40Crossref PubMed Scopus (15) Google Scholar). These data are consistent with the conclusion that residues along ACP helix α2, such as Glu-41 and Glu-48, are important for ACP function, although the binding of ACP to individual components in the crude extracts used in this work was not addressed. One surprise that emerged from this body of work was the importance of Ile-54 for ACP function. Ile-54 lies in the loop region between helices α2 and α3, and whether this highly conserved amino acid has a role in docking or acyl chain binding is unknown. Somers and co-workers (10Parris K.D. Lin L. Tam A. Mathew R. Hixon J. Stahl M. Fritz C.C. Seehra J. Somers W.S. Structure. 2000; 8: 883-895Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) determined the crystal structure of ACP from Bacillus subtilis in complex with AcpS. The contacts between ACP and AcpS are predominantly ionic occurring between the positively charged residues of AcpS helix α1 and the negatively charged residues of ACP helix α2. The hydrophobic residues on ACP helix α2 (Met-44 and Ala-45) are complemented by hydrophobic counterparts on AcpS. Thus, the AcpS-ACP structure provides direct support for the hypothesis that the conserved helix α2 (“recognition helix”) of ACP functions as a universal protein interaction domain (11Zhang Y.-M. Marrakchi H. White S.W. Rock C.O. J. Lipid Res. 2003; 44: 1-10Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This study explores the recognition helix hypothesis with a second enzyme of type II fatty acid synthesis by examining the protein-protein interaction from the standpoint of both the enzyme and ACP. The fabG gene product, β-ketoacyl-ACP reductase, was selected because it is an essential and universally expressed component of type II fatty acid biosynthesis. FabG catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP intermediates, and only a single isoform is known. In addition, the crystal structures of E. coli FabG (16Price A.C. Zhang Y.-M. Rock C.O. White S.W. Biochemistry. 2001; 40: 12772-12781Crossref PubMed Scopus (133) Google Scholar) and the Brassica napas FabG·NADP+ binary complex (17Fisher M. Kroon J.T. Martindale W. Stuitje A.R. Slabas A.R. Rafferty J.B. Structure. 2000; 8: 339-347Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) facilitate the analysis of the FabG surface adjacent to the entrance to the active site. We used site-directed mutagenesis to determine the residues on FabG required for high affinity ACP binding and protein NMR spectroscopy to identify the ACP residues that contribute to the formation of the FabG·ACP complex. Materials—Amersham Biosciences supplied the [1-14C]acetyl-CoA; Sigma supplied the acetyl-CoA, malonyl-CoA, and ACP; Qiagen supplied the Ni2+-nitrilotriacetic acid resin; Promega supplied the molecular biology reagents. NH2-terminally His-tagged FabD, FabH, and FabG were expressed in E. coli strain BL21(DE3) (Novagen) and purified by nickel chelation affinity chromatography as described previously (18Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 19Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Protein was stored in 50% glycerol at -20 °C. Protein concentration was determined using the Bradford method with γ-globulin as the standard (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). All other reagents were of the highest available purity. Construction of FabG and ACP Mutants—Mutations were introduced into the fabG gene by using an overlap extension PCR method. Wild type fabG gene was cloned into pET-15b plasmid (pET-fabG) using NdeI and BamHI sites. All of the FabG mutants were prepared using the same two outside primers on the plasmid, T7 promoter primer (5′-TAATACGACTCACTATAGGG) and T7 terminator primer (5′-GCTAGTTATTGCTCAGCGG). The internal primers for all the FabG mutants are listed in Table I. To construct each mutant, two PCRs with the wide type plasmid as the template, one outside primer and the respective inside primer were performed, and the products were then pooled and used as a template for a second PCR using both outside primers. The ∼980-bp PCR product was purified from a 1% agarose gel using QIAquick gel extraction kit (Qiagen) and ligated into pCR2.1 (Invitrogen). Following transformation into E. coli INVαF′, plasmid DNA was isolated and sequenced. A clone with the sequence containing the desired mutation was digested with NdeI and BamHI and ligated into pET-15b, which had been digested with the same enzymes and dephosphorylated with calf intestinal alkaline phosphorylase. The resultant plasmids were transformed into competent E. coli BL21(DE3) cells by electroporation. Expression and purification was as for the wild type protein. The proper folding of the mutant proteins was checked by circular dichroism spectroscopy.Table IPrimers used for FabG mutagenesisMutantForward primeraCodons changed to introduce the mutations are underlined.Reverse primerArg 129 → Ala5′-GAAAAAGGCTCATGGTCGTATTATCAC5′-ACGACCATGAGCCTTTTTCATCATAGArg 129 → Glu5′-GAAAAAGGAACATGGTCGTATTATCAC5′-ACGACCATGTTCCTTTTTCATCATAGArg 172 → Ala5′-GCGTCAGCCGGTATTACTGTAAAC5′-AATACCGGCTGACGCAACTTCGArg 172 → Glu5′-GCGTCAGAAGGTATTACTGTAAAC5′-AATACCTTCTGACGCAACTTCGa Codons changed to introduce the mutations are underlined. Open table in a new tab ACP-dependent Gel Reconstitution Assay of FabG Activity—FabG activity was tested in an ACP-dependent assay by analyzing the formation of β-hydroxybutyryl-ACP with a conformational sensitive gel electrophoresis method. The assay contained 25 μm ACP, 1 mm β-mercaptoethanol, 65 μm malonyl-CoA, 45 μm [1-14C]acetyl-CoA (specific activity 60 μCi/μmol), 200 μm NADPH, 1 μg of purified FabD, 0.5 μg of purified FabH in 0.1 m sodium phosphate buffer, pH 7.0, and 1 ng to 0.256 μg of FabG (or FabG mutant) protein in a final volume of 40 μl. The ACP, β-mercaptoethanol, and buffer were preincubated at 37 °C for 30 min to ensure the complete reduction of ACP. The substrate for the FabG reaction was generated using FabD to transfer the malonyl group from CoA to ACP to produce malonyl-ACP and FabH to condense acetyl-CoA and malonyl-ACP to form β-ketobutyryl-ACP. The reaction was initiated by the addition of FabG. After incubation at 37 °C for 15 min, the reaction was stopped by adding 8 μl of 6× sample buffer to the reaction mix and placing the reaction tubes on ice. Then 40 μl of each of the assay mixtures was loaded onto conformational sensitive gels that contained 13% acrylamide and 0.5 m urea. The electrophoresis was performed under constant current of 32 mA/gel at 25 °C. The gels were stained in Coomassie Blue, destained, and dried with a vacuum gel drier at 80 °C. The gels were exposed against PhosphorImager screens that were scanned by Typhoon 9200. The product of FabG, β-hydroxy-[1-14C]butyryl-ACP, was quantitated with ImageQuant 5.2. ACP-independent Spectrophotometric Assay of FabG Activity—This assay measured the disappearance of NADPH, the cofactor for FabG reaction, spectrophotometrically at 340 nm. The reaction mixture contained 0.5 mm acetoacetyl-CoA, 0.2 mm NADPH, 1-20 μg of FabG (or FabG mutant) protein, 0.1 m sodium phosphate buffer, pH 7.4, in a final volume of 300 μl. The reaction was initiated by the addition of acetoacetyl-CoA. Decrease in the absorbance at 340 nm was recorded for 2 min. The initial rate was used to calculate the enzymatic activity. The ability of ACP to inhibit either FabG or FabG mutant activity in the spectrophotometric assay was tested by incubating ACP with the protein at room temperature for 5 min before the addition of acetoacetyl-CoA to initiate the reaction. FabG-ACP Binding Measured by Surface Plasmon Resonance—Binding studies were performed on a Biacore 3000 Surface Plasmon Resonance instrument. ACP was covalently attached to a carboxymethyl-dextran-coated gold surface (CM-5 Chip, Biacore). The carboxymethyl groups on the chip were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to activate the carboxymethyl-dextran. The ACP was attached at pH 4.5 to this activated surface by reaction of the carboxyl groups of the dextran with primary amines on the ACP to form an amide linkage. The remaining reactive sites were blocked by reaction with ethanolamine. A reference cell was prepared similarly except that no ACP was added. Binding was measured by flowing 370 nm FabG in 10 mm HEPES, 150 mm NaCl, pH 7.4, at a flow rate of 20 μl/min through the reference and ACP-containing flow cells in sequence. A blank run consisted of buffer only. Following the injection, release of the bound FabG was measured by flowing only buffer through the flow cells. Regeneration of the chip surface to remove bound FabG consisted of allowing the protein to dissociate in buffer for 60 min between injections. The data reported show the difference in surface plasmon resonance signal between the flow cell containing ACP and the reference cell. Mutant FabG concentrations used in the assay were 335 nm for FabG[R172A] and 408 nm for FabG[R172E]. FabG-ACP Binding Measured by AlphaScreen—AlphaScreen technology is an approach to study biological interactions by energy transfer (22Beaudet L. Bedard J. Breton B. Mercuri R.J. Budarf M.L. Genome Res. 2001; 11: 600-608Crossref PubMed Scopus (56) Google Scholar). Upon laser excitation, a chemical signal is generated on the donor beads (streptavidin-coated). When a specific interaction brings the acceptor beads (a specific antibody coated) to the proximity of the donor beads, a cascade of energy transfers take place, emitting highly amplified fluorescence (or AlphaScreen signal) at a wavelength that is lower than that of the excitation. Binding between the biotinylated ACP and His-tagged FabG brings streptavidin donor beads and nickel chelate acceptor beads together, emitting AlphaScreen signals. ACP was biotinylated using EZ-Link™ sulfo-NHS-LC-biotin (Pierce). To study the ACP binding ability of FabG and FabG mutants, 1 μm biotinylated ACP was incubated with different concentrations (30 nm to 2 μm) of His-tagged FabG at room temperature in a 384-well ProxiPlate (Packard Instrument Co.). After a 30-min incubation, streptavidin donor beads and nickel chelate acceptor beads (50 μg/ml final concentration for each bead, (His)6Tag detection system, Packard Instrument Co.) were added to the above solutions. The reaction mixtures were then incubated for 1 h before being read by Fusion™ Universal Microplate Analyzer (Packard Instrument Co.), with excitation at 680 nm and emission at 600 nm. NMR Spectroscopy and Titration Experiments—All NMR data were acquired with Varian Inova 600-MHz spectrometer at 27 °C. Data were processed and displayed by the program package NMRPipe and NMRDraw (23Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11280) Google Scholar) on an SGI Octane work station. The programs XEASY (ETH Automated Spectroscopy for X Window System) and Sparky (24Goddard T.D. Kneller D.G. SPARKY 3.0. University of California, San Francisco1998Google Scholar) were used for data analysis. Backbone resonance of 13C/15N-labeled ACP was determined on the basis of three-dimensional HNCO, HNCACB, CBCA(CO)NH (25Yamazaki T. Lee W. Arrowsmith D.H. Muhandriam D. Kay L.E. J. Am. Chem. Soc. 1994; 116: 11655-11666Crossref Scopus (491) Google Scholar), and HSQC-TOCSY (26Marion D. Driscoll P.C. Kay L.E. Wingfield P.T. Bax A. Gronenborn A.M. Clore G.M. Biochemistry. 1989; 28: 6150-6156Crossref PubMed Scopus (930) Google Scholar). Titrations of FabG to 15N-ACP were monitored by 15N-1H HSQC-TROSY experiments. The molar ratios of FabG:ACP at each titration point were 0.07, 0.14, 0.21, 0.28, 0.35, 0.42, 0.54, 0.65, and 0.85, respectively. For the first six points in the titration series of 15N-labeled ACP with FabG, 50 μl of 0.96 mm FabG in 40 mm potassium phosphate, pH 6.5, 10 mm EDTA, 1 mm dithiothreitol were added successively to the NMR tube containing 550 μl of 1.34 mm15N-labeled ACP in the same buffer. The samples were mixed thoroughly after each addition. For the last two points, 80 μl of 0.96 mm FabG was added. When the mutant FabG[R129E/R172E] was titrated to 15N-labeled ACP, 70 μl of 0.93 mm mutant FabG in the same buffer as wild type FabG was added successively to 460 μl of 1.34 mm15N-labeled ACP. The molar ratios of FabG[R129E/R172E]:ACP in the titration series were 0.11, 0.22, 0.33, 0.43, 0.54, and 0.65, respectively. The Electropositive/Hydrophobic Patch at the Entrance to the FabG Active Site—FabG is a highly conserved and universal component of the type II system that catalyzes the NADPH-dependent reduction of all β-ketoacyl-ACP intermediates in the elongation cycle. Unlike other enzymes in the fatty acid biosynthetic pathway, there is only a single FabG isozyme that has been identified (2Jackowski S. Rock C.O. Biochem. Biophys. Res. Commun. 2002; 292: 1155-1166Crossref PubMed Scopus (162) Google Scholar). Multiple sequence alignment indicates that FabG sequences from different species including bacteria and plants share a high degree of sequence identity (Fig. 1A). The crystal structures of FabG from E. coli (16Price A.C. Zhang Y.-M. Rock C.O. White S.W. Biochemistry. 2001; 40: 12772-12781Crossref PubMed Scopus (133) Google Scholar) and B. napus (17Fisher M. Kroon J.T. Martindale W. Stuitje A.R. Slabas A.R. Rafferty J.B. Structure. 2000; 8: 339-347Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) are also homologous and reveal FabG as a tetramer consisting of dimers composed of monomers arranged in a head-to-tail configuration (Fig. 1B). Two conserved arginine residues, Arg-129 and Arg-172, are located adjacent to the active site opening (Fig. 1). We have shown previously (12Zhang Y.-M. Rao M.S. Heath R.J. Price A.C. Olson A.J. Rock C.O. White S.W. J. Biol. Chem. 2001; 276: 8231-8238Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) that the positively charged residues at the entrance to the FabH active site are important for the binding of the ACP substrate. The location of the conserved arginines within a hydrophobic area adjacent to the entrance to the FabG active site tunnel suggested they were key residues in the interaction with the acidic residues located along helix α2 of ACP. A Crucial Role for Arg-129 and Arg-172 of FabG for ACP Binding—FabG mutants of Arg-129 and Arg-172 were generated by site-directed mutagenesis. Two assays were used to evaluate the effects of these mutations on the FabG-specific activity. The first was ACP-dependent and used β-ketobutyryl-ACP as the substrate in a radiochemical assay (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 27Tsay J.-T. Oh W. Larson T.J. Jackowski S. Rock C.O. J. Biol. Chem. 1992; 267: 6807-6814Abstract Full Text PDF PubMed Google Scholar). The second assay was ACP-independent. β-Ketobutyryl-CoA was used as a substrate analog in place of β-ketobutyryl-ACP, and the disappearance of NADPH was monitored spectrophotometrically at 340 nm. The wild type FabG enzyme was much less active with the β-ketobutyryl-CoA analog compared with its normal substrate β-ketobutyryl-ACP (Table II). Mutations that selectively interfere with the FabG-ACP interaction adversely affect the ACP-dependent assay but not the ACP-independent assay. All FabG mutants had circular dichroism spectra that were identical to the wild type enzyme (not shown) indicating that the mutations did not perturb the overall protein structure.Table IISpecific activity of FabG wild type and the ACP-binding site mutants in the ACP-dependent and ACP-independent assaysFabGSpecific activity (% activity)ACP-dependentACP-independentμmol/min/μgμmol/min/mgWild type2.90 ± 0.49 (100%)2.60 ± 0.22 (100)R129E0.245 ± 0.006 (8.5)2.68 ± 0.082 (103)R129A0.284 ± 0.013 (9.8)3.24 ± 0.059 (124.5)R172E0.102 ± 0.011 (3.5)1.04 ± 0.027 (40.0)R172A0.405 ± 0.042 (14.0)1.60 ± 0.041 (61.5)R129E/R172E0.029 ± 0.006 (1.0)1.47 ± 0.017 (56.5)R129A/R172A0.110 ± 0.011 (3.8)2.08 ± 0.023 (80.0) Open table in a new tab Table II lists the FabG mutants that were produced and their activities in the two assays. All of the FabG mutant proteins exhibited similar activities with the CoA substrate analog compared with the wild type FabG. This result illustrated that the FabG mutants were catalytically competent, a finding anticipated because the surface arginine mutations were not directed against the known active site residues buried in the active site cavity of FabG (16Price A.C. Zhang Y.-M. Rock C.O. White S.W. Biochemistry. 2001; 40: 12772-12781Crossref PubMed Scopus (133) Google Scholar, 28Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In contrast, all of the mutant proteins exhibited significantly reduced enzyme activity with β-ketobutyryl-ACP in comparison to the wild type enzyme (Table II). Changing the arginine residues to glutamate was more deleterious than changing the residues to the neutral alanine. The most affected protein was the FabG[R129E/R172E] double mutant. These biochemical data are consistent with the idea that these two arginines are important for the interaction between ACP and FabG. Thus, this biochemical analysis showed that the panel of FabG mutants were selectively deficient in the utilization of ACP thioester substrates suggesting a defect in FabG-ACP interaction. The binding of ACP to FabG and the panel of mutant proteins was directly addressed using AlphaScreen™ technology described under “Experimental Procedures.” Biotinylated ACP and His-tagged FabG, either wild type or the individual mutants, were incubated together before the addition of the streptavidin-coated donor beads and the nickel-chelate acceptor beads. Interactions between FabG and ACP bring the two beads into proximity, which allows an energy transfer cascade to take place following laser excitation that leads to the production of a fluorescent signal that was detected by a fusion reader. Wild type FabG exhibited strong AlphaScreen signals at less than 1 μm concentration (Fig. 2). In contrast, neither the single mutants nor the double mutants produced a signal even at higher protein concentrations, illustrating that mutations at the two Arg residues of FabG attenuated its ability to bind ACP. To corroborate the binding results obtained with AlphaScreen™, two other methods were used to directly measure the FabG-ACP interaction. Wild type FabG and the Arg-172 single mutants were used in these experiments. The first approach used was surface plasmon resonance (BIAcore®). In these experiments (see “Experimental Procedures” and Ref. 16Price A.C. Zhang Y.-M. Rock C.O. White S.W. Biochemistry. 2001; 40: 12772-12781Crossref PubMed Scopus (133) Google Scholar), the ACP molecule was covalently attached to the chip (the “ligand”), and FabG flowed across the chip and was monitored for binding (the “analyte”). The important results are shown in Fig. 3. Wild type FabG produced a robust signal at 370 nm, whereas with FabG[R172E] and FabG[R172A] there was little evidence for ACP binding at similar protein concentrations. The apparent dissociation constant for the FabG-ACP interaction calculated in this study was 4.7 μm using the on and off rate constants determined in the plasmon resonance experiment. This number was essentially the same as the 4.5 μm dissociation constant reported by our group earlier (16Price A.C. Zhang Y.-M. Rock C.O. White S.W. Biochemistry. 2001; 40: 12772-12781Crossref PubMed Scopus (133) Google Scholar), where a more comprehensive surface plasmon resonance analysis of ACP binding to wild type FabG is presented. The second method was based on the ability of free ACP to inhibit the FabG reaction using β-ketobutyryl-CoA substrate analog (the ACP-independent assay described under “Experimental Procedures”). When bound to FabG, ACP and its prosthetic group occludes the active site tunnel and prevents the entry of β-ketobutyryl-CoA. This effect will be attenuated in FabG mutants that have reduced affinity for ACP. The results of these experiments using wild type FabG, FabG[R172E], and FabG[R172A] are presented in Fig. 4. ACP exhibited an IC50 of 120 μm with the wild type enzyme, but both FabG[R172E] and FabG[R172A] were refractory to ACP inhibition. These data coupled with the direct binding experiments provide strong evidence that A
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