Roles of the Active Site Water, Histidine 303, and Phenylalanine 396 in the Catalytic Mechanism of the Elongation Condensing Enzyme of Streptococcus pneumoniae
2006; Elsevier BV; Volume: 281; Issue: 25 Linguagem: Inglês
10.1074/jbc.m513199200
ISSN1083-351X
AutoresYongmei Zhang, Jason C. Hurlbert, Stephen W. White, Charles O. Rock,
Tópico(s)Genomics and Phylogenetic Studies
Resumoβ-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (β-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nϵ stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nϵ of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes. β-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (β-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nϵ stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nϵ of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes. The condensing enzymes play a central role in fatty acid biosynthesis by elongating the growing acyl chain by two carbon atoms to initiate each elongation cycle (1Jackowski S. Rock C.O. Biochem. Biophys. Res. Commun. 2002; 292: 1155-1166Crossref PubMed Scopus (172) Google Scholar, 2Smith S. Witkowski A. Joshi A.K. Prog. Lipid Res. 2003; 42: 289-317Crossref PubMed Scopus (498) Google Scholar). Somewhat uniquely in biological synthesis, the enzymes create a carbon-carbon bond via a Claisen-like condensation reaction (3Heath R.J. Rock C.O. Nat. Prod. Rep. 2002; 19: 581-596Crossref PubMed Scopus (194) Google Scholar). Specifically, they catalyze the condensation of malonyl-acyl carrier protein (ACP) 2The abbreviations used are: ACP, acyl carrier protein; CoA, coenzyme A; TAL, triacetic acid lactone; DTT, dithiothreitol. with an acyl-ACP intermediate via a two-step ping-pong kinetic mechanism. In the first step, an acyl chain from either acyl-CoA or acyl-ACP is transferred to an active site cysteine, and the cofactor is released. During the second step, malonyl-ACP binds, and a carbanion is generated on the C2 of malonate concomitant with the release of the C3 carboxyl group (4Greenspan M.D. Alberts A.W. Vagelos P.R. J. Biol. Chem. 1969; 244: 6477-6485Abstract Full Text PDF PubMed Google Scholar, 5Vagelos P.R. Alberts A.W. J. Biol. Chem. 1960; 235: 2786-2791Abstract Full Text PDF PubMed Google Scholar, 6Witkowski A. Joshi A.K. Smith S. Biochemistry. 2002; 41: 10877-10887Crossref PubMed Scopus (65) Google Scholar). The carbanion then attacks the acyl-enzyme intermediate to produce the β-ketoacyl-ACP product. In the dissociated, type II synthases, the condensation reaction is carried out by monofunctional enzymes (7Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar), and most bacteria have only a single elongation-condensing enzyme that belongs to the FabF class. In mammals and yeast, the condensing enzyme component, KS, is fused into a multidomain complex referred to as the type I or associated FAS system (8Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar). However, it is clear from primary sequence analysis that the active site of the FAS I condensation module is very similar to the FAS II elongation enzymes (Fig. 1A). The polyketide synthases also contain a condensing enzyme module in which the same signature active site residues can be identified (Fig. 1A). The importance of the elongation condensing enzymes in regulating fatty acid formation (7Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 9Cronan Jr., J.E. Rock C.O. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1996; (American Society for Microbiology, Washington, D. C.): 612-636Google Scholar) and the unique chemistry of the reaction that they catalyze (3Heath R.J. Rock C.O. Nat. Prod. Rep. 2002; 19: 581-596Crossref PubMed Scopus (194) Google Scholar) have focused our interest on understanding the specific tasks of each active site residue in catalysis. In addition, these enzymes have emerged as attractive targets for the development of new broad-spectrum antibiotics (10Heath R.J. White S.W. Rock C.O. Prog. Lipid Res. 2001; 40: 467-497Crossref PubMed Scopus (294) Google Scholar, 11Campbell J.W. Cronan Jr., J.E. Annu. Rev. Microbiol. 2001; 55: 305-332Crossref PubMed Scopus (420) Google Scholar, 12Heath R.J. Rock C.O. Curr. Opin. Investig. Drugs. 2004; 5: 146-153PubMed Google Scholar) and anti-obesity/anti-cancer drugs (13Slade R.F. Hunt D.A. Pochet M.M. Venema V.J. Hennigar R.A. Anticancer Res. 2003; 23: 1235-1243PubMed Google Scholar, 14Gao S. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5628-5633Crossref PubMed Scopus (84) Google Scholar, 15Takahashi K.A. Smart J.L. Liu H. Cone R.D. Endocrinology. 2004; 145: 184-193Crossref PubMed Scopus (40) Google Scholar, 16Menendez J.A. Vellon L. Mehmi I. Oza B.P. Ropero S. Colomer R. Lupu R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10715-10720Crossref PubMed Scopus (288) Google Scholar, 17Cha S.H. Hu Z. Lane M.D. Biochem. Biophys. Res. Commun. 2004; 317: 301-308Crossref PubMed Scopus (59) Google Scholar), and there is growing interest in engineering the polyketide synthases to produce novel therapeutic agents (18Khosla C. Tang Y. Science. 2005; 308: 367-368Crossref PubMed Scopus (16) Google Scholar). These efforts will be facilitated by a complete mechanistic understanding of the active site. At their catalytic cores, the elongation enzymes possess a Cys-His-His triad. These residues have been mutated and are thought to be critical to the overall forward condensation reaction (8Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar, 19Huang W. Jia J. Edwards P. Dehesh K. Schneider G. Lindqvist Y. EMBO J. 1998; 17: 1183-1191Crossref PubMed Scopus (179) Google Scholar, 20Moche M. Dehesh K. Edwards P. Lindqvist Y. J. Mol. Biol. 2001; 305: 491-503Crossref PubMed Scopus (59) Google Scholar, 21Price A.C. Rock C.O. White S.W. J. Bacteriol. 2003; 185: 4136-4143Crossref PubMed Scopus (47) Google Scholar, 22Amy C.M. Witkowski A. Naggert J. Williams B. Randhawa Z. Smith S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3114-3118Crossref PubMed Scopus (134) Google Scholar), although the functions of these residues in the partial reactions of the catalytic cycle are not precisely known. There are also conserved lysine, glutamate and phenylalanine residues in the vicinity of the active site, and the structural and/or catalytic roles for these residues are not understood (Fig. 1A). Our systematic analysis of a panel of mutants in a model elongation condensing enzyme and the correlation of this new information with new and previously determined structures led to a reevaluation of the roles for the active site residues in the catalytic cycle. We selected SpFabF as the basis for this work, since its x-ray structure is known at the highest resolution (1.3 Å) (21Price A.C. Rock C.O. White S.W. J. Bacteriol. 2003; 185: 4136-4143Crossref PubMed Scopus (47) Google Scholar). A stereo view of the SpFabF active site illustrating the orientation of the key residues investigated in this study is shown in Fig. 1B. Materials—Myristoyl-ACP was prepared using an established acyl-ACP synthetase method (23Rock C.O. Garwin J.L. J. Biol. Chem. 1979; 254: 7123-7128Abstract Full Text PDF PubMed Google Scholar, 24Rock C.O. Garwin J.L. Cronan Jr., J.E. Methods Enzymol. 1981; 72: 397-403Crossref PubMed Scopus (44) Google Scholar). [2-14C]Malonyl-CoA and sodium [14C]bicarbonate were purchased from Amersham Biosciences. [1-14C]Lauroyl-CoA was purchased from American Radiolabeled Chemicals. Malonyl-ACP was prepared using AcpS (ACP synthase) with apo-ACP and malonyl-CoA as described (25Zhang L. Joshi A.K. Hofmann J. Schweizer E. Smith S. J. Biol. Chem. 2005; (M413686200)Google Scholar). His-tagged AcpS, FabD, FabG, FabI, FabZ, FabB (β-ketoacyl-ACP synthase I), FabF, and FabH (β-ketoacyl-ACP synthase III) from Escherichia coli and FabF from Streptococcus pneumoniae were purified as described previously (21Price A.C. Rock C.O. White S.W. J. Bacteriol. 2003; 185: 4136-4143Crossref PubMed Scopus (47) Google Scholar, 26Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 27Price A.C. Choi K.H. Heath R.J. Li Z. Rock C.O. White S.W. J. Biol. Chem. 2001; 276: 6551-6559Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Proteins were quantitated by the Bradford method (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Construction of Mutants and Protein Purification—A DNA fragment encoding the wild-type S. pneumoniae FabF was cloned into the pET15b vector between the NdeI and BamHI sites. Mutations at the active site residues were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All clones were checked by automated sequencing on an ABI Prism 3700 DNA Analyzer. His-tagged mutant proteins were expressed in Rossetta (DE3) cells and purified with Ni2+-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA). The proper folding of the mutant proteins was confirmed by circular dichroism spectroscopy. β-Ketoacyl-ACP Synthase Assay—The condensation assay for EcFabB and SpFabF was described previously (29Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11956Abstract Full Text PDF PubMed Google Scholar). Briefly, the assays contained 45 μm myristoyl-ACP, 50 μm [2-14C]malonyl-CoA (specific activity, 52 Ci/mol), 100 μm ACP, 1 μg of EcFabD, and the indicated amount of EcFabB or SpFabF in a final volume of 20 μl. ACP was reduced by 0.3 mm DTT before the other reaction components were added. After incubation of the sample at 37 °C for 20 min, the reaction was stopped by adding 0.4 ml of reducing agent containing 0.1 m K2HPO4, 0.4 m KCl, 30% tetrahydrofuran, and 5 mg/ml NaBH4. The reaction mixtures were vigorously mixed and incubated at 37 °C for 40 min, followed by extraction with 0.4 ml of toluene. The 14C-isotope in the upper phase was quantitated. Reconstituted Fatty Acid Synthase Assay—The overall fatty acid synthase activity in vitro was measured using purified fatty acid biosynthetic enzymes (30Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 1833-1836Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The 40-μl reaction contained 200 μm ACP, 200 μm NADH, 200 μm NADPH, 50 μm acetyl-CoA, 250 μm [2-14C]malonyl-CoA (specific activity, 10.4 mCi/mmol), 2 μg of EcFabD, 2 μg of EcFabH, 2 μg of EcFabG, 2 μg of EcFabZ, 2 μg of EcFabI, and 4 μg wild-type or mutant SpFabF in 100 mm sodium phosphate buffer, pH 7. ACP was reduced by 1 mm β-mecaptoethanol for 20 min at 37 °C before the remaining components were added. Following incubation at 37 °C for 30 min, the reactions were stopped by placing the reaction tubes on an ice slurry. The entire mixture was loaded onto a conformationally sensitive 2 m urea, 13% acrylamide gel, and the gel was stained, dried, and exposed to a phosphor storage screen (Amersham Biosciences). The radioactivity was detected by a Typhoon 9200 PhosphoImager and quantitated using ImageQuant software. Transacylation Assay—The assay for the transacylase activity of the condensing enzyme contained 25 μm ACP, 0.3 mm DTT, 50 μm [1-14C]lauroyl-CoA (specific activity, 55 mCi/mmol) and the indicated amount of (2, 1, 0.5, 0.25, or 0.125 μg) enzyme in 100 mm potassium phosphate, pH 6.8, buffer in a total volume of 40 μl. ACP was reduced by DTT in buffer before the rest of the reaction components were added. Following incubation at 37 °C for 30 min, the reactions were stopped by pipetting 35 μl of the reaction mix onto DE81 filter discs (Whatman). The discs were then washed three times with chloroform/methanol/acetic acid (2:5:3 volume ratio) containing 0.2 m LiCl (20 min/wash with 20 ml/disc). The discs were dried and counted to quantitate [14C]acyl-ACP. Malonyl-ACP Decarboxylation Assay—The decarboxylase assay measured the conversion of malonyl-ACP to acetyl-ACP and triacetic acid lactone (TAL) (31Zha W. Shao Z. Frost J.W. Zhao H. J. Am. Chem. Soc. 2004; 126: 4534-4535Crossref PubMed Scopus (45) Google Scholar). The reaction mix contained 50 μm ACP, 0.3 mm DTT, 50 μm [2-14C]malonyl-CoA (specific activity, 52 mCi/mmol), 1 μg of EcFabD, and 10 μg of wild-type or mutant SpFabF in 100 mm potassium phosphate, pH 6.8, in a final volume of 60 μl (26Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). ACP was reduced by DTT in buffer before the rest of the reaction components were added. The reaction was started by the addition of SpFabF. Following incubation at 37 °C for 40 min, the reactions were stopped by placing the reaction tubes on an ice slurry, and 35- and 25-μl aliquots were removed. The 35-μl portion was loaded onto a conformationally sensitive 0.5 m urea, 13% acrylamide gel. The 25-μl reaction mix was spotted on a preadsorbent Silica Gel G plate (Analtech) developed in chloroform/methanol/acetic acid (24:1:1, v/v/v), dried, and exposed to a phosphor storage imager (Amersham Biosciences). The distribution of radioactivity was detected and quantitated as described above. Malonyl-ACP Bicarbonate Exchange Reaction—The carbon exchange between malonyl-ACP and bicarbonate is the reverse reaction of the condensing enzyme (5Vagelos P.R. Alberts A.W. J. Biol. Chem. 1960; 235: 2786-2791Abstract Full Text PDF PubMed Google Scholar). The reaction contained 50 μm malonyl-ACP, 5 μm myristoyl-ACP, 10 mm [14C]bicarbonate and 8 μg of wild-type SpFabF or mutants in 100 mm potassium phosphate, pH 6.8, in a final volume of 20 μl. Following incubation at 37 °C for 20 min, the reaction was stopped by placing the tubes in an ice slurry before being loaded onto a conformationally sensitive 0.5 m urea, 13% acrylamide gel. Radioactivity was detected and quantitated as described above. Complementation of the fabB(Ts) Growth Phenotype—Strain CY274 (fabB15(Ts)) was unable to grow at the nonpermissive temperature (42 °C) unless unsaturated fatty acids (oleate) were supplied or the fabB gene was expressed in trans. The ability of wild-type and mutant FabB to complement the fabB(Ts) phenotype was tested after transformation of strain CY274 with plasmids carrying wild-type or mutant fabB (32Tsay J.-T. Rock C.O. Jackowski S. J. Bacteriol. 1992; 174: 508-513Crossref PubMed Google Scholar). SpFabF-ACP Interactions—AlphaScreen technology is an experimental method to quantitate protein-protein interactions (33Beaudet L. Bedard J. Breton B. Mercuri R.J. Budarf M.L. Genome Res. 2001; 11: 600-608Crossref PubMed Scopus (57) Google Scholar). Upon laser excitation, a chemical signal is generated on the donor beads (streptavidin-coated). When a specific interaction brings the acceptor beads (coated with a specific antibody or nickel chelater) 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. We measured ACP affinity using the competitive binding approach. Briefly, 1 μm biotinylated ACP was mixed with 1 μm His-tagged wild-type SpFabF or mutant in the presence of different concentrations of regular ACP (120 nm to 10 μm) in a 384-well ProxiPlate (PerkinElmer Life Sciences). After a 30-min incubation, streptavidin donor beads and Ni2+ chelate acceptor beads (50 μg/ml final concentration for each bead; Alphascreen Histidine Detection Kit, PerkinElmer Life Sciences) were added to the above solutions. The reaction mixtures were then incubated for 1 h before being read by Fusion™ Universal Microplate Analyzer (PerkinElmer Life Sciences), with excitation at 680 nm and emission at 600 nm. X-ray Crystallography—Crystals of the SpFabF[H303A] mutant were grown in 20% polyethylene glycol 3350, 0.2 m potassium acetate and were in space group P21212 with cell dimensions a = 60.39 Å, b = 88.82 Å, and c = 61.05 Å. Rod-shaped crystals measuring ∼0.25 mm on edge were harvested after 9 days and frozen in liquid nitrogen using 25% PEG 400 as a cryoprotectant. A 2.6 Å data set was collected at the SER-CAT ID beamline (Sector 22 at the Advanced Photon Source in Argonne National Laboratory), and processed using MOSFLM (34Leslie A.G.W. Joint CCP4 and ESF-EAMCB Newsletter on Protein Crystallography. 1992; 26Google Scholar) and SCALA (35Evans P.R. Joint CCP4 and ESF-EAMCB Newsletter on Protein Crystallography. 1997; 33: 22-24Google Scholar). The structure was solved by molecular replacement, using the program AMoRe (36Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 869-873Crossref PubMed Scopus (5030) Google Scholar) and the native SpFabF structure (Protein Data Bank code 1OX0) as the search model, and refined using a combination of REFMAC (37Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) and CNS (38Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Data collection and refinement statistics are shown in Table 3. Model building was carried out using the O program (39Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar).TABLE 3Statistics of data collection and refinementParameter (Unit)SpFabF[H303A]Space groupP21212Unit cell dimensions (Å) (a, b, c)60.4, 88.8, 61.0Resolution range (Å)61.1 to 2.6RsymaValues in parentheses refer to the highest resolution shell,bRsym = ΣΣ|Ii–Im|/ΣΣIi, where Ii is the intensity of the measured reflection, and Im is the mean intensity of all symmetry-related reflections0.046 (0.472)I/σaValues in parentheses refer to the highest resolution shell37.1 (3.5)Completeness (%)99.5RedundancyaValues in parentheses refer to the highest resolution shell4.7 (4.7)Reflections254,362Unique reflections10,590Resolution range included in refinement (Å)30 to 2.6No. of reflections in working set10,506No. of reflections in test set (5%)500No. of protein atoms in ASU3090No. of water molecules ASU40Rwork0.194Rtest0.256Root mean square deviation from ideal stereochemistry Bond lengths (Å)0.007 Bond angles (degrees)1.315Mean B overall (Å2)34.75Ramachandran plot Most favored region (%)86.9 Additionally allowed region (%)11.9 Generously allowed (%)0.6 Disallowed (%)0.6a Values in parentheses refer to the highest resolution shellb Rsym = ΣΣ|Ii–Im|/ΣΣIi, where Ii is the intensity of the measured reflection, and Im is the mean intensity of all symmetry-related reflections Open table in a new tab Condensing Enzyme Activity of FabF Mutants—A subset of completed conserved residues within four protein segments in the elongation class of condensing enzymes of FAS I, FAS II, and the polyketide synthases was selected for analysis (Fig. 1). The active site cysteine (Cys164) and the two histidines (His303 and His337) that comprise the central Cys-His-His triad were obvious choices for site-directed mutagenesis. Also selected were Lys332 and Glu346 that have been implicated in catalysis, although Glu346 is not conserved in FAS I and polyketide synthases (Fig. 1). Finally, Phe396 was also included in the analysis, because it is located in a flexible region of the active site surrounded by conserved glycines. Each of these residues (Fig. 1) was mutated to alanine in SpFabF, and the mutants were analyzed for their ability to carry out the biochemical activities that comprise the full and partial reactions of the condensing enzyme catalytic cycle. The overall condensation reaction was determined using [2-14C]malonyl-ACP and myristoyl-ACP as substrates. With the exception of SpFabF[H303A] and SpFabF[E346A], which retained 26 and 100% of the wild-type activity, respectively, all of the mutant condensing enzymes were deficient in their overall condensation activity (Fig. 2A, Table 1). The function of the mutants was also evaluated in an assay to reconstitute multiple rounds of fatty acid synthesis starting with acetyl-CoA as the primer (Fig. 2B). The SpFabF[H303A] and SpFabF[F396A] mutants retained activity, whereas the other mutants were inactive in this assay format, although neither active mutant produced as much long-chain acyl-ACPs as the wild type. In order to detect very low levels of activity, the assays were repeated at higher protein concentrations, and this revealed that SpFabF[K332A] exhibited a low, but significant, activity that was estimated at 0.2% of wild-type (Table 1). The activity of the other mutant proteins was not detected at the highest protein concentrations permitted by the assay format, setting the condensation activity of these constructs to <0.2% (Table 1).TABLE 1Summary of the activities of S. pneumoniae FabF active site mutants The specific activities for the 100% reactions were as follows: condensation, 147 ± 7 pmol/μg/min; transacylation, 4.15 ± 0.46 pmol/μg/min; acetyl-ACP formation, 2.49 ± 0.04 pmol/μg/min; and TAL formation, 0.122 ± 0.03 pmol/μg/min.SpFabFCondensation Myristoyl-ACP + [2-14C]Mal-ACP ↓ 14C-β-ketopalmitoyl-ACPTransacylation 14C-Lauroyl-CoA + ACP ↓ 14C-lauroyl-ACPDecarboxylationAc-ACP formation [2-14C]Mal-ACP ↓ HCO3– + 14C-Ac-ACPTAL formation 3 [2-14C]Mal-ACP ↓ 3 HCO3– + 14C-TAL%%%%Wild-type100100–a<0.2% of control activity100C164A100–K332A0.228.53.510H303A25.660–500H337A–122.5–C164A/K332A––3.4–C164A/H303A––100–C164A/H337A––2.5–F396A0.759.44.339.9E346A100100–100a <0.2% of control activity Open table in a new tab The results with SpFabF[H303A] and SpFabF[K332A] were inconsistent with previous conclusions from work on EcFabB (40McGuire K.A. Siggaard-Andersen M. Bangera M.G. Olsen J.G. Wettstein-Knowles P. Biochemistry. 2001; 40: 9836-9845Crossref PubMed Scopus (49) Google Scholar). This study concluded that the equivalent residues of His303 and Lys332 in EcFabB are both essential, whereas our analysis indicated that the these two residues have important, but not absolutely essential, roles in promoting the overall condensation reaction. This apparent inconsistency might be attributable to subtle differences between the FabB and FabF subfamilies. We explored this point by constructing the same panel of mutants in EcFabB as reported previously (40McGuire K.A. Siggaard-Andersen M. Bangera M.G. Olsen J.G. Wettstein-Knowles P. Biochemistry. 2001; 40: 9836-9845Crossref PubMed Scopus (49) Google Scholar); all of the FabB mutants appeared to be inactive using a protein concentration in the linear range for the wild-type enzyme (not shown). However, we were able to detect a trace of activity (∼1% of wild type) in the EcFabB[H298A] and EcFabB[K328A] mutants when assayed at higher protein concentrations, illustrating that they retained partial condensation activity in vitro. We corroborated this conclusion using an in vivo complementation test to verify the activities of the FabB mutants (Table 2). The panel of fabB mutant alleles was introduced into E. coli strain CY274 (fabB15(Ts)), and the resulting strains were scored for correction of the temperature-sensitive growth phenotype (Table 2). The EcFabB[H298A] and EcFabB[K328A] mutants restored growth at the nonpermissive temperature, demonstrating that they retained condensing enzyme activity in vivo. In contrast, the inactive EcFabB[C163A] and EcFabB[H333A] mutants were unable to complement the growth phenotype. Importantly, since the condensation enzymes function as dimers, the lack of activity of the latter two mutants ruled out the possibility that complementation arose from the stabilization of the temperature-sensitive fabB allele by the expression of a properly folded protein. The results with the E. coli mutant panel (Table 2) were qualitatively the same as with the SpFabF panel (Table 1), although the level of enzyme activity in the EcFabB[H298A] was less than the residual activity in SpFabF[H303A]. Thus, the different condensing enzyme mutant sets differed in their absolute values of activity, but the rank order of activity in the mutant proteins was the same.TABLE 2Complementation of E. coli strain CY274 (fabB15(Ts)) by a panel of fabB mutantsPlasmidGrowth of strain CY27430 °C42 °CpBluescript+–pfabB++pfabB[C163A]+–pfabB[K328A]++pfabB[H298A]++pfabB[H333A]+– Open table in a new tab Active Site Residues Participating in Transacylation—The first step in the condensing enzyme reaction is the transfer of the acyl group to the active site cysteine, and this activity was assayed by monitoring the transfer of the isotopic labeled acyl group from [1-14C]lauroyl-CoA to ACP via the acyl-enzyme intermediate. As expected, mutants lacking this critical cysteine were completely inactive in the transacylation step (Fig. 2C). In contrast, SpFabF[H337A], SpFabF[H303A], SpFabF[F396A], SpFabF[E346A], and SpFabF[K332A] all retained transacylation activity (Fig. 2C, Table 1), indicating that none of these residues were absolutely essential for transthioesterification. The transacylation assay can also detect accumulation of the [14C]acyl-enzyme intermediate, and this was only measurable for the SpFabF[K332A] mutant (not shown), indicating the removal of the conserved lysine either accelerated the rate of formation or reduced the rate of release of the acyl-enzyme intermediate. These results showed that the only active site residue required for transacylation was Cys164. The finding that the SpFabF[H337A] mutant had 12% the transacylation activity of wild-type (4.15 ± 0.46 pmol/μg/min) suggests that it has little influence on the ionization of the active site cysteine. Although His337 is within hydrogen-bonding distance of Cys164, its electronic state is inconsistent with a role for this residue in deprotonating the active site sulfhydryl. The Nδ nitrogen of His337 accepts a hydrogen bond from a backbone amide, meaning that the lone pair cannot be on the Nϵ nitrogen adjacent to Cys164 (Fig. 1B). Thus, the fixed electronic state of the histidine renders it unable to facilitate the abstraction of a proton from Cys164. Perhaps one could postulate a conformational change, but none of the many available structures give any hint of this. The cerulenin and thiolactomycin inhibitor structures (27Price A.C. Choi K.H. Heath R.J. Li Z. Rock C.O. White S.W. J. Biol. Chem. 2001; 276: 6551-6559Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 41Moche M. Schneider G. Edwards P. Dehesh K. Lindqvist Y. J. Biol. Chem. 1999; 274: 6031-6034Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) show that the Nϵ nitrogen of His337 donates a hydrogen bond to the carbonyl of the antibiotics, supporting the placement of the lone pair on Nδ rather than on the Nϵ adjacent to Cys164. In the related FabH condensing enzymes, this histidine is replaced by an asparagine residue, and the Oδ1 receives the same hydrogen bond from the backbone amide as does the Nδ nitrogen of His337, again placing the protonated nitrogen adjacent to the active site cysteine. Although there may be mechanistic differences between the FabH and FabB/F classes of condensing enzymes, the requirement to generate the thiolate is a common feature and is attributed to the strong helix dipole conserved between the two proteins rather than the histidine, which is not conserved (26Davies C. Heath R.J. White S.W. Rock C.O. Structure. 2000; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Residues Participating in the Decarboxylation Step—The formation of acetyl-ACP from malonyl-ACP measures the ability of the enzymes to create the carbanion intermediate via the release of the C3 carboxyl group and occurs in the absence of an acyl-enzyme intermediate. Although this is a futile, nonproductive reaction, it independently measures the status of the decarboxylation half-reaction. This partial reaction requ
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