Title: A New Mechanism for Anaerobic Unsaturated Fatty Acid Formation inStreptococcus pneumoniae
Abstract: The anaerobic pathway for unsaturated fatty acid synthesis was established in the 1960s in Escherichia coli. The double bond is introduced into the growing acyl chain by FabA, an enzyme capable of both the dehydration of β-hydroxydecanoyl-acyl carrier protein (ACP) to trans-2-decenoyl-ACP, and the isomerization of trans-2 to cis-3-decenoyl-ACP. However, there are a number of anaerobic bacteria whose genomes do not contain a fabA homolog, although these organisms nonetheless produce unsaturated fatty acids. We cloned and biochemically characterized a new enzyme in type II fatty acid synthesis from Streptococcus pneumoniae that carries out the isomerization of trans-2-decenoyl-ACP tocis-3-decenoyl-ACP, but is not capable of catalyzing the dehydration of β-hydroxy intermediates. This tetrameric enzyme, designated FabM, has no similarity to FabA, but rather is a member of the hydratase/isomerase superfamily. Thus, the branch point in the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation oftrans-2-decenoyl-ACP, in contrast to E. coliwhere the branch point takes place after the formation of β-hydroxydecanoyl-ACP. The anaerobic pathway for unsaturated fatty acid synthesis was established in the 1960s in Escherichia coli. The double bond is introduced into the growing acyl chain by FabA, an enzyme capable of both the dehydration of β-hydroxydecanoyl-acyl carrier protein (ACP) to trans-2-decenoyl-ACP, and the isomerization of trans-2 to cis-3-decenoyl-ACP. However, there are a number of anaerobic bacteria whose genomes do not contain a fabA homolog, although these organisms nonetheless produce unsaturated fatty acids. We cloned and biochemically characterized a new enzyme in type II fatty acid synthesis from Streptococcus pneumoniae that carries out the isomerization of trans-2-decenoyl-ACP tocis-3-decenoyl-ACP, but is not capable of catalyzing the dehydration of β-hydroxy intermediates. This tetrameric enzyme, designated FabM, has no similarity to FabA, but rather is a member of the hydratase/isomerase superfamily. Thus, the branch point in the biosynthesis of unsaturated fatty acids in S. pneumoniae occurs following the formation oftrans-2-decenoyl-ACP, in contrast to E. coliwhere the branch point takes place after the formation of β-hydroxydecanoyl-ACP. Unsaturated fatty acid (UFA) 1The abbreviations used are: UFA, unsaturated fatty acid; ACP, acyl carrier protein; SFA, saturated fatty acids; FabA, β-hydroxydecanoyl-ACP dehydratase/isomerase; FabB, β-ketoacyl-ACP synthase I; FabF, β-ketoacyl-ACP synthase II; FabH, β-ketoacyl-ACP synthase III; FabI, enoyl-ACP reductase I; FabK, enoyl-ACP reductase II; FabZ, β-hydroxyacyl-ACP dehydratase; FabD, malonyl-CoA:ACP transacylase; NAC, N-acetylcysteamine; ec, E. coli; sp, S. pneumoniae; mt, M. tuberculosis; ca, C. acetobutylicum. biosynthesis is essential for the maintenance of membrane structure and function in many groups of bacteria that embrace the anaerobic life style. In eukaryotes, olefin formation requires molecular oxygen (1Bloomfied D. Bloch K. J. Biol. Chem. 1960; 235: 337-345Google Scholar), and double bonds are introduced into the fatty acids following the completion of their synthesis via the type I, multifunctional fatty acid synthase (2Smith S. FASEB J. 1994; 8: 1248-1259Google Scholar). In contrast, bacteria synthesize fatty acids using the dissociated, type II fatty acid synthase system in which each of the steps is catalyzed by distinct enzymes that are encoded by separate genes (3Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtis R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D. C.1996: 612-636Google Scholar, 4Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Google Scholar). The key players in UFA synthesis were first defined by the isolation and characterization of UFA-auxotrophs (5Clark D.P. Cronan Jr., J.E. Methods Enzymol. 1981; 72: 693-707Google Scholar). In the type II system, the double bond is introduced anaerobically into the growing acyl chain at the 10-carbon intermediate by β-hydroxydecanoyl-ACP dehydratase, FabA (6Kass L.R. Bloch K. Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 1168-1173Google Scholar). FabA is capable of both the removal of water to generate trans-2-decenoyl-ACP and the isomerization of this intermediate to the cis-3-decenoyl-ACP (3Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtis R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D. C.1996: 612-636Google Scholar, 7Bloch K. Acc. Chem. Res. 1968; 2: 193-202Google Scholar). However, FabA is not the only protein that is required for introduction of the double bond and does not catalyze the rate-limiting step in UFA formation (8Clark D.P. de Mendoza D. Polacco M.L. Cronan Jr., J.E. Biochemistry. 1983; 22: 5897-5902Google Scholar). A second unsaturated fatty acid auxotroph was isolated that corresponds to the fabB gene, which encodes β-ketoacyl-ACP synthase I. In fabB mutants, saturated fatty acid synthesis persists due to the presence of the other elongation condensing enzyme inEscherichia coli, FabF (9D'Agnolo G. Rosenfeld I.S. Vagelos P.R. J. Biol. Chem. 1975; 250: 5289-5294Google Scholar, 10Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11956Google Scholar). Although FabF readily elongates 16:1 to 18:1 (10Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11956Google Scholar), the inability to support UFA synthesis infabB mutants leads to the conclusion that FabF cannot elongate a key intermediate in UFA biosynthesis (3Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtis R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D. C.1996: 612-636Google Scholar, 4Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Google Scholar). The analysis offabB and fabF mutants, coupled with the catalytic properties of FabB and FabF in vitro supports a function for FabB in UFA synthesis and a role for FabF in the thermal modulation of membrane fatty acid composition (11Gelmann E.P. Cronan Jr., J.E. J. Bacteriol. 1972; 112: 381-387Google Scholar, 12Garwin J.L. Cronan Jr., J.E. J. Bacteriol. 1980; 141: 1457-1459Google Scholar, 13Ulrich A.K. de Mendoza D. Garwin J.L. Cronan Jr., J.E. J. Bacteriol. 1983; 154: 221-230Google Scholar, 14de Mendoza D. Cronan Jr., J.E. Trends Biochem. Sci. 1983; 8: 49-52Google Scholar). The availability of numerous bacterial genomes sequences allows the reconstruction of type II fatty acid synthase in these organisms using standard bioinformatics analysis tools. It is notable thatfabA and fabB genes occur together in most bacteria that produce UFA (15Campbell J.W. Cronan Jr., J.E. J. Bacteriol. 2001; 183: 5982-5990Google Scholar). However, many anaerobes that synthesize UFA, such as the Streptococci and Clostridia, do not have a recognizable fabA homolog in their genomes, and also have afabF rather than a fabB subtype of elongation condensing enzyme. Clearly, UFA are synthesized by a distinct biochemical mechanism in these organisms, and the goal of this study was to identify the enzyme(s) responsible for olefin formation inStreptococcus pneumoniae. Like E. coli, S. pneumoniae produces straight-chain saturated and monounsaturated fatty acids predominately of 16 and 18 carbon chain lengths (16Trombe M.C. Laneelle M.A. Laneelle G. Biochim. Biophys. Acta. 1979; 574: 290-300Google Scholar). Our experiments show that this organism does not utilize a FabA-like mechanism for introducing a double bond into the growing acyl chain, but rather accomplishes this task using a previously unknown enzyme, termed trans-2, cis-3-decenoyl-ACP isomerase (FabM). Reconstitution of the S. pneumoniae UFA synthetic pathway in vitro and in vivo lead to the conclusion that the branch point for UFA synthesis occurs at the enoyl-ACP intermediate, and the amount of UFA produced arises from the competition of FabM and FabK for enoyl-ACP. Sources of supplies were: Amersham Biosciences, [2-14C]malonyl-CoA (specific activity, 56 mCi/mmol); Sigma, antibiotics, acyl-CoA, ACP; Difco, microbiological media; Promega, molecular reagents and restriction enzymes; Invitrogen, T4 ligase; Novagen, pET vectors and expression strains; Qiagen, Ni2+-agarose resin. Protein was quantitated by the Bradford method (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). The Mycobacterium tuberculosis mtFabH, E. coli ecFabD, ecFabG, ecFabA, S. pneumoniae spFabZ, and spFabF proteins were purified as described previously (18Choi K.-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Google Scholar, 19Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Google Scholar, 20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar, 21Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Google Scholar, 22Schujman G.E. Choi K.-H. Altabe S. Rock C.O. de Mendoza D. J. Bacteriol. 2001; 183: 3032-3040Google Scholar). All other chemicals were reagent grade or better. The fabM gene was amplified from genomic DNA from S. pneumoniae R6. The spfabM PCR primer pair consisted of 5′-AAATAAAAAGGAGCCCATATG and 5′-GGATCCTCAAAGAATGATGCAAG. The primers introduced novel restriction sites for NdeI at the initiator methionine codon of the predicted coding sequence and BamHI downstream of the stop codon. The PCR products were ligated into the plasmid pCR2.1 and sequenced to verify the absence of PCR mutations. The plasmid was isolated and digested with NdeI andBamHI, and the gene fragment was isolated and ligated into plasmid pET-15b digested with the same enzymes. The resulting plasmid was used to transform strain BL21(DE3) codonplus-RIL strain, and the protein was overexpressed and purified as described previously (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar). Affinity chromatography was followed by gel filtration on Superdex-200 HR 26/60. The enzyme was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of FabM was estimated by gel filtration chromatography using a Superdex-200 HR10/30 column calibrated with globular protein standards. Cycles of fatty acid elongation were reconstituted in vitro to detect isomerase activity using the purified individual enzymes that catalyze the fatty acid biosynthesis cycle essentially as described previously (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar, 23Choi K.-H. Heath R.J. Rock C.O. J. Bacteriol. 2000; 182: 365-370Google Scholar). The reaction mixtures contained 100 μm ACP, 10 mm dithiothreitol, 0.1 m sodium phosphate buffer, pH 7.0, 100 μm NADPH, 100 μm NADH, 50 μm octanoyl-CoA, 100 μm[14C]malonyl-CoA (specific activity, 56 mCi/mmol), mtFabH (1.0 μg/reaction), ecFabD (1.0 μg), ecFabG (1.0 μg), spFabZ (2.3 μg/reaction), ecFabA (2 μg/reaction), spFabF (3 μg) or spFabM (5 μg) in a final volume of 40 μl. The assay mixtures were incubated at 37 °C for 20 min and analyzed by conformationally sensitive gel electrophoresis in 15% polyacrylamide gels containing 2.5m urea. Electrophoresis was performed at 25 °C and 32 mA/gel. The gels were dried, and the bands were quantitated using a phosphorimager screen. Specific activities were calculated from the slopes of the plot of product formation versus protein concentration in the assay. Bands were identified based on the generation of standards as described previously (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar, 21Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Google Scholar). The substrate specificity of FabM was addressed by substituting 100 μmeither decanoyl-CoA, lauroyl-CoA, or myristoyl-CoA for octanoyl-CoA in the assay. This assay measures spectrophotometrically the conversion oftrans-2-octenoyl-NAC to cis-3-octenoyl-NAC. A solution containing 100 μm trans-2-octenoyl-NAC in 10 mm potassium phosphate, pH 7.0, was placed in a cuvette, and the substrate concentration was verified by determining the absorbance at 263 nm (ε = 6700 m−1cm−1) (24Seubert W. Lynen F. J. Am. Chem. Soc. 1953; 75: 2787Google Scholar). The reaction was started by the addition of the enzyme to 150-μl final volume, and the decrease in absorbance at 263 nm was followed on a Shimadzu UV-visible spectrophotometer UV-1601. Each reaction was run in triplicate. One unit is defined as the disappearance of 1 pmol oftrans-2-octenoyl-NAC per min under the defined conditions. Specific activity was expressed as units per microgram of protein. The solutions of acyl-ACP derivatives from the reconstituted assays described above were adjusted to 2% acetic acid, and the protein mass determinations were performed by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. Approximately 100 pmol of the protein were diluted to 50 μl with 2% acetonitrile, 1% acetic acid. The protein was loaded on a C8 reversed phase nano-extraction cartridge (Western Analytical, Murrieta, CA) and washed extensively with the same buffer to remove bound salts. The protein was eluted from the column with 30 μl of 80% acetonitrile, 1% acetic acid and then diluted with 1% acetic acid to a final acetonitrile concentration of 50%. Mass measurements were performed using an LCT electrospray-time of flight spectrometer (Micromass Inc, Beverly, MA) equipped with a Z-spray electrospray interface (Micromass Inc, Beverly, MA). A flow rate of 10 μl/min was maintained using a VLP200 syringe pump (Harvard Apparatus, Holliston, MA), and the desalted protein was introduced by loop injection. Data were collected for an m/zrange of 500–2500 at a cone voltage of 35 V and a manual pusher time of 70 μs. All other instrument settings are those typically used for protein measurements on this instrument. Deconvolution of the protein spectrum was accomplished using the maximum entropy algorithm of the MassLynx software (Micromass Inc, Beverly, MA) (25Ferrige A.G. Seddon M.J. Green B.N. Jarvis S.A. Skilling J. Rapid Commun. Mass. Spectrom. 1992; 6: 707-711Google Scholar). Strain JT60 (fabA(Ts)) was unable to grow at the non-permissive temperature (42 °C) unless unsaturated fatty acids (oleate) were supplied or the fabA gene expressed (26Rock C.O. Tsay J.T. Heath R. Jackowski S. J. Bacteriol. 1996; 178: 5382-5387Google Scholar). The ability of spFabM alone or in conjunction with caFabK to complement thefabA(Ts) phenotype was tested after transformation of JT60 with plasmids carrying either spfabM, cafabK, or both. The same vector (pBluescript KSII(+)) and construction method were used to constitutively express the His-tagged versions of spFabM, caFabK, and ecFabA (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar). The Clostridium acetobutylicum cafabK gene (CAC3576) was amplified from genomic DNA from the ATCC strain 824. The cafabK PCR primers were: 5′-CATATGTTAAAAACTCAGTTTTGTG and 5′-GGATCCCCTATTTAATTCTATCTATAACT. The primers introduced restriction sites for NdeI at the initiator methionine and BamHI downstream of the stop codon. The caFabK protein was expressed from pBluescript and found to substitute for all of the spFabK functions. Like plasmids expressingspfabK (27Heath R.J. Rock C.O. Nature. 2000; 406: 145-146Google Scholar), cafabK expression restored growth of an E. coli fabI(Ts) mutant at 42 °C, illustrating its function as an enoyl reductase in type II fatty acid synthesis in vivo. Expression of caFabK also conferred triclosan resistance to E. coli. In order to select for the presence of both FabK and FabM, pBluescript vector bearing thecafabK was modified to replace the AmpR gene with a KanR cassette using AvaII andDraIII restriction sites. The pBluescript (empty vector), pfabA (positive control), pfabM, pfabK, or a combination of pfabK/pfabM, pfabK/pfabA were transformed into JT60 and selected with the appropriate antibiotic combinations. Cells were grown at the permissive temperature for the host strain (30 °C), and individual colonies were spotted onto rich broth agar plates without or with 0.15 μg/ml triclosan and incubated at 42 °C. Plates were scored for growth after 24 h at 42 °C. An analysis of the type II fatty acid biosynthetic genes in S. pneumoniae show that they cluster at a single location within the genome (Fig.1). A comparison of the predicted protein sequences of these open reading frames to the known enzymes of E. coli showed that the S. pneumoniae gene cluster lacked both FabI and FabA homologs. Recently, the open reading frame termed fabK (Fig. 1) was demonstrated to encode a novel flavoprotein enoyl-ACP reductase that replaces FabI in theS. pneumoniae type II system (27Heath R.J. Rock C.O. Nature. 2000; 406: 145-146Google Scholar). There are two unknown genes at the end of the cluster of known fatty acid biosynthetic genes. One gene, SP0416, is predicted to encode a helix-turn-helix DNA binding protein of the MarR family that may be a transcriptional regulator involved in controlling the expression of this gene cluster. Adjacent to the transcription factor is the SP0415 open reading frame that is renamed in this work as fabM. Further bioinformatics analysis of the gene cluster reveals a potential connection between the fabM, HTH (SP0416), andfabK genes. The MarR transcription factor is a dimer that utilizes a winged-helix motif to bind a DNA palindrome (28Alekshun M.N. Kim Y.S. Levy S.B. Mol. Microbiol. 2000; 35: 1394-1404Google Scholar, 29Alekshun M.N. Levy S.B. Mealy T.R. Seaton B.A. Head J.F. Nat. Struct. Biol. 2001; 8: 710-714Google Scholar). Often bacterial transcription factors are autoregulated, and their DNA binding motifs are located within their own promoter regions. A DNA palindrome was located in the promoter region of the putative SP0416 transcriptional regulator (Fig. 1). Significantly, this same sequence palindrome is found in the promoters of the fabM andfabK genes (Fig. 1). Unraveling the transcriptional regulation in this large cluster is beyond the scope of this study. The significance of the bioinfomatic analysis is that it tiesfabM to the fatty acid biosynthetic gene cluster and suggests that the fabM and fabK genes may be coordinately regulated. The fabM open reading frame specifies a protein that is a member of the hydratase/isomerase superfamily (Pfam 000378, Ref. 30Bateman A. Birney E. Durbin R. Eddy S.R. Howe K.L. Sonnhammer E.L. Nucleic Acids Res. 2000; 28: 263-266Google Scholar). A comparison of the predicted protein sequence to three members of this superfamily and to the family consensus sequence is illustrated in Fig.2. FabM has a strong similarity to the consensus sequence of Pfam 000378 (Fig. 2) exhibiting 28% identity over the 169-amino acid sequence. This family of enzymes catalyze a wide variety of reactions centered on double bond isomerizations and water addition and elimination at the α,β carbons of thioester substrates. The structures of family members show a common active site design that provides for CoA binding, an expandable acyl chain binding pocket, an oxyanion hole for polarizing the thioester carbonyl, and multiple active site stations for the positioning of acidic and basic amino acid side chains to facilitate proton shuffling (31Xiang H. Luo L. Taylor K.L. Dunaway-Mariano D. Biochemistry. 1999; 38: 7638-7652Google Scholar). The SP0415 (fabM) open reading frame is annotated in the data base as an enoyl-CoA hydratase/isomerase. The hydratase/isomerase activity that the data base entry is referring to is associated with the enzymes responsible for either hydrating enoyl-CoA to β-hydroxyacyl-CoA or isomerizing cis-3 to trans-2-enoyl-CoA intermediates in fatty acid β-oxidation. However, S. pneumoniae lacks cytochromes and does not possess enzymes of the β-oxidation pathway making it highly unlikely that FabM functions in this context (32Tettelin H. Nelson K.E. Paulsen I.T. Eisen J.A. Read T.D. Peterson S. Heidelberg J. DeBoy R.T. Haft D.H. Dodson R.J. Durkin A.S. Gwinn M. Kolonay J.F. Nelson W.C. Peterson J.D. Umayam L.A. White O. Salzberg S.L. Lewis M.R. Radune D. Holtzapple E. Khouri H. Wolf A.M. Utterback T.R. Hansen C.L. McDonald L.A. Feldblyum T.V. Angiuoli S. Dickinson T. Hickey E.K. Holt I.E. Loftus B.J. Yang F. Smith H.O. Venter J.C. Dougherty B.A. Morrison D.A. Hollingshead S.K. Fraser C.M. Science. 2001; 293: 498-506Google Scholar). PhaB (PaaB) is an enzyme essential for the catabolism of phenylacetic acid in Pseudomonas putida and is thought to carry out either the hydroxylation or isomerization of the double bonds once the aromatic ring has been opened (33Olivera E.R. Minambres B. Garcia B. Muniz C. Moreno M.A. Ferrandez A. Diaz E. Garcia J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Google Scholar). This protein is part of a multifunctional phenylacetic acid degradation complex and shows 35% identity and 51% similarity to FabM. ChcB is a novel Δ3,Δ2-enoyl-CoA isomerase involved in the biosynthesis of the cyclohexanecarboxylic acid moiety of the polyketide ansatrienin A (34Patton S.M. Cropp T.A. Reynolds K.A. Biochemistry. 2000; 39: 7595-7604Google Scholar). ChcB has 27% identity and 41% similarity to FabM. FadB is a multifunctional protein involved in fatty acid β-oxidation inE. coli, and contains as one of its activities acis-3-trans-2-enoyl-CoA isomerase (35Yang S.Y. Li J.M. He X.Y. Cosloy S.D. Schulz H. J. Bacteriol. 1988; 170: 2543-2548Google Scholar). The section of this protein that aligns with FabM is the component of the complex thought to be responsible for the enoyl-CoA isomerase activity required in the degradation of unsaturated fatty acids and over this segment of the protein has a 30% identity and 46% similarity to FabM. These strong similarities to enzymes known to catalyze isomerizations of enoyl thioesters led us to test the hypothesis that FabM encodes atrans-2 to cis-3-decenoyl-ACP isomerase. The FabM open reading frame was cloned into pET-15b, and the His-tagged fusion protein purified by affinity chromatography and gel filtration as described under “Experimental Procedures.” The purified protein has a monomeric molecular size of 31 kDa (Fig.3). Members of the hydratase/isomerase protein family are uniformly multimeric proteins with hexamers of identical subunits being a common configuration, although some members are dimers and tetramers. We therefore examined the size of native FabM by gel filtration chromatography to estimate its aggregation state (Fig. 3). These results are consistent with FabM existing as a tetramer of identical subunits in solution. The ability of FabM to act as an isomerase was tested in a reconstituted fatty acid biosynthetic system designed to detect isomerase activity (Fig.4). Cycles of fatty acid elongation were reconstituted in vitro using purified enzymes as described under “Experimental Procedures.” The assay employed the FabH enzyme from M. tuberculosis to generate β-keto[14C]decanoyl-ACP starting with octanoyl-CoA and [2-14C]malonyl-ACP (via ecFabD) as substrates. The NADPH-dependent ecFabG reduced the intermediate to the initial substrate for the assays, β-hydroxy[14C]decanoyl-ACP (Fig.4 A,lane 1). The addition of ecFabA (lane 2) results in the conversion of the β-hydroxy intermediate to a mixture of trans-2- and cis-3-decenoyl-ACPs. These isomeric forms are not distinguished on the gel, but previous work suggests that the trans intermediate would predominate (36Schwab J.M. Klassen J.B. Lin D.C.T. Anal. Biochem. 1985; 150: 121-124Google Scholar). The enoyl-ACP cannot be elongated by a condensing enzyme, but thecis-3 intermediate can. Accordingly, the addition of spFabF, the elongation condensing enzyme of S. pneumoniae, condenses the cis-3-decenoyl-ACP with malonyl-ACP, and following reduction by ecFabG, gives rise to the accumulation of a new band on the gel corresponding to β-hydroxy-cis-5-dodecanoyl-ACP (Fig. 4 A,lane 3). Since this reaction mixture did not contain an enoyl-ACP reductase, additional rounds of elongation cannot occur, and the product accumulates at the 12-carbon stage. Furthermore, ecFabA is characteristically inactive with unsaturated β-hydroxy intermediates (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar), so there is little conversion of the β-hydroxy-cis-5-dodecenoyl-ACP to the trans-2 intermediate. The addition of spFabZ also converts the β-hydroxydecanoyl-ACP to the enoyl-ACP (Fig. 4 A,lane 4); however, the addition of spFabF to this reaction did not lead to the appearance of the elongated 12-carbon unsaturated intermediate (Fig. 4, lane 5). These data illustrate that spFabZ is a dehydratase and that it is not capable of isomerizing thetrans-2-enoyl-ACP to the cis-3 intermediate. The addition of FabM to the base reaction did not lead to the formation of enoyl-ACP (Fig. 4 A,lane 6), and in combination with spFabF did not lead to the appearance of any additional products (lane 7). Thus, FabM lacked β-hydroxyacyl-ACP dehydratase activity. The combination of spFabZ and FabM led to the formation of enoyl-ACP (lane 8), but it was not possible to discern if the cis intermediate was formed in this reaction mixture. In the presence of spFabZ, spFabF, and FabM, a new product appeared indicating that cycles of elongation occurred (Fig.4 A,lane 9). This product(s) arises from the elongation of the cis-3 intermediate by spFabF and the dehydration by spFabZ. In contrast to FabA and like ecFabZ (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar), spFabZ was capable of utilizing unsaturated β-hydroxy intermediates to form enoyl-ACPs. These experiments with the reconstituted fatty acid synthase enzymes establish that FabM is capable of isomerizingtrans-2-enoyl-ACP to cis-3-acyl-ACP, but cannot dehydrate β-hydroxyacyl-ACP. In addition, spFabF is capable of elongating cis-3-acyl-ACP intermediates. The reconstituted fatty acid synthase assay is a crude tool for detailed biochemical characterization of an individual enzyme specificity, but we employed this assay to evaluate the substrate specificity of FabM isomerase (Fig. 4, panel B). The mtFabH/FabG/spFabZ system was used to present FabM with different chain length enoyl-ACP substrates. The formation of an elongation product arising from FabM isomerase activity as a function of FabM protein in the assay was used to estimate the activity of FabM. FabM was most active when the 10-carbon enoyl-ACP was presented as the substrate exhibiting a specific activity under these defined conditions of 1.6 ± 0.09 pmol/min/μg. The 12-carbon enoyl-ACP was also utilized in vitro, albeit at a much lower rate (0.4 ± 0.08 pmol/min/μg). We examined 14- and 16-carbon enoyl-ACPs as substrate; however, there was no evidence for isomerization of these longer substrates (not shown). The low activity of mtFabH with hexanoyl-CoA (18Choi K.-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Google Scholar) did not permit the analysis of FabM activity ontrans-2-octenoyl-ACP using this assay. The specific activity of ecFabA for the 10-carbon substrate under these same assay conditions was 33 ± 1 pmol/min/μg. Thus, FabM was 20-fold less efficient than ecFabA in the formation of cis-double bonds in thein vitro fatty acid synthase assay reconstituted with the indicated constellation of enzymes and the E. coli ACP cofactor. These data are consistent with FabM, like ecFabA (20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Google Scholar, 37Endo K. Helmkamp Jr., G.M. Bloch K. J. Biol. Chem. 1970; 245: 4293-4296Google Scholar), being most active on 10-carbon enoyl-ACP, but capable of isomerizing longer chain substrates at a lower rate. The ACP thioester intermediates in the reconstitution assays (Fig. 4 A) were analyzed by electrospray ionization mass spectrometry (ESI-MS) to confirm the identities of the products (Fig.5). The major mass peak for the ACP starting material occurred at 8849 with a minor peak at mass 8980 corresponding to the ACP molecules that retained the amino-terminal methionine residue. A new mass peak of 9019 with the expected mass increase of 170 appeared in the β-hydroxydecanoyl-ACP starting material (lane 1). The trans-2-andcis-3-decenoyl-ACP mixture (lane 2) formed after dehydration by ecFabA led to the appearance of a new peak at mass 9001 (ACP+152). The elongated product of cis-3-decenoyl-ACP by spFabF was predicted to be the β-hydroxy-cis-5-dodecenoyl-ACP (lane 3), and accordingly the mass spectrum of the mixture contained a new peak at 9044 corresponding to ACP+196. Samples from the reactions inlanes 4 and 5 containedtrans-2-decenoyl-ACP and displayed the expected mass peak at 9001. The β-hydroxydecanoyl-ACP precursor (lanes 6 and7) was not converted to other products by FabM, and the predominant acyl-ACP peak in these reactions mixtures was 9019, diagnostic for β-hydroxydecanoyl-ACP (Fig. 5 A). Inlane 8, trans-2- and/orcis-3-decenoyl-ACPs were revealed by the appearance of a characteristic mass peak at 9001. The FabM-catalyzed isomerization of the trans-2 to cis-3-decenoyl-ACP was revealed by the appearance of acyl-ACP products elongated by spFabF (lane 9). Two products were detected as illustrated by the appearance of a peak for cis-5, trans-2-C12:2-ACP (ACP+178) as well as cis-7,cis-5,trans-2-C14:3-ACP (ACP+204) with predicted and observed molecular masses of 9027 and 9053, respectively (Fig.5 B). 2Fatty acid abbrevi