Domain Swapping between Enterococcus faecalis FabN and FabZ Proteins Localizes the Structural Determinants for Isomerase Activity
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m504637200
ISSN1083-351X
AutoresYing‐Jie Lu, Stephen W. White, Charles O. Rock,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoAnaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of β-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489–34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the β3 and β4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of β-hydroxyacyl-ACP dehydratases is determined by the properties of the β-sheets that dictate the orientation of the central α-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site. Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of β-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489–34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the β3 and β4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of β-hydroxyacyl-ACP dehydratases is determined by the properties of the β-sheets that dictate the orientation of the central α-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site. Anaerobic unsaturated fatty acid (UFA) 1The abbreviations used are: UFA, unsaturated fatty acid; ACP, acyl carrier protein; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; FabA, β-hydroxydecanoyl-ACP dehydratase/isomerase; FabN, FabZ-like protein of E. faecalis with β-hydroxydecanoyl-ACP dehydratase/isomerase activity; FabZ, β-hydroxyacyl-ACP dehydratase. Species-specific proteins are designated with the genus-species abbreviation in italics in front of the protein name such as: EcFabZ for E. coli FabZ and EfFabN for E. faecalis FabN. biosynthesis occurs through the insertion of a double bond into the growing acyl chain (Fig. 1), and the proportion of UFA produced is a critical determinant of the biophysical properties of biological membranes. In bacteria, fatty acids are synthesized using the dissociated, type II fatty acid synthase system in which each of the steps is catalyzed by distinct enzymes that are each encoded by separate genes (1Cronan 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, 2Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (294) Google Scholar). The key players in UFA synthesis in Escherichia coli were first defined by the isolation and characterization of UFA auxotrophs (3Clark D.P. Cronan Jr., J.E. Methods Enzymol. 1981; 72: 693-707Crossref PubMed Scopus (11) Google Scholar). The double bond is introduced at the 10-carbon intermediate by β-hydroxydecanoyl-ACP dehydratase, FabA (4Kass L.R. Bloch K. Proc. Natl. Acad. Sci. U. S. A. 1967; 58: 1168-1173Crossref PubMed Scopus (62) Google Scholar), which 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 (1Cronan 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, 5Bloch K. Acc. Chem. Res. 1968; 2: 193-202Crossref Scopus (147) Google Scholar) (Fig. 1). A second UFA auxotroph was isolated which corresponds to the fabB gene, which encodes β-ketoacyl-ACP synthase I (6Clark D.P. de Mendoza D. Polacco M.L. Cronan Jr., J.E. Biochemistry. 1983; 22: 5897-5902Crossref PubMed Scopus (60) Google Scholar). In fabA and fabB mutants, saturated fatty acid synthesis persists because of the presence of another dehydratase, FabZ (7Mohan S. Kelly T.M. Eveland S.S. Raetz C.R.H. Anderson M.S. J. Biol. Chem. 1994; 269: 32896-32903Abstract Full Text PDF PubMed Google Scholar), and another elongation-condensing enzyme, FabF (8D'Agnolo G. Rosenfeld I.S. Vagelos P.R. J. Biol. Chem. 1975; 250: 5289-5294Abstract Full Text PDF PubMed Google Scholar, 9Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11956Abstract Full Text PDF PubMed Google Scholar). The FabZ isozyme (β-hydroxyacyl-ACP dehydratase) is expressed ubiquitously in type II systems, cannot carry out the isomerase reaction, and is the only type of dehydratase that exists in most bacteria. Although FabA and FabZ have many primary sequence characteristics in common, bioinformatic analysis clearly divide the two subtypes. Specifically, there are distinct differences in the active site residues, an Asp in FabA and a Glu in FabZ, and FabA is a dimer (10Leesong M. Henderson B.S. Gillig J.R. Schwab J.M. Smith J.L. Structure. 1996; 4: 253-264Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) whereas FabZ is a hexamer (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). FabZ does play a role in UFA synthesis by functioning in the elongation of both saturated and unsaturated long chain acyl-ACP, whereas FabA most efficiently processes saturated chain lengths 10 carbons and shorter (12Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Likewise, the FabF condensing readily elongates 16:1 to 18:1 UFA (9Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11956Abstract Full Text PDF PubMed Google Scholar); however, the inability to support UFA synthesis in fabB mutants leads to the conclusion that FabF cannot elongate a key intermediate in UFA biosynthesis in vivo, most likely cis-3-decenoyl-ACP (1Cronan 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, 2Rock C.O. Cronan Jr., J.E. Biochim. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (294) Google Scholar). The availability of numerous bacterial genome sequences allows the reconstruction of type II fatty acid synthesis in these organisms using bioinformatics analysis tools. It is notable that fabA and fabB genes occur together in Gram-negative bacteria that produce UFA (13Campbell J.W. Cronan Jr., J.E. J. Bacteriol. 2001; 183: 5982-5990Crossref PubMed Scopus (101) Google Scholar). However, many anaerobes that synthesize UFA do not have a recognizable fabA homolog in their genomes and also have a FabF rather than a FabB subtype of elongation-condensing enzyme. In these organisms UFA is synthesized by a different mechanism. Streptococcus pneumoniae produces straight chain saturated and monounsaturated fatty acids predominantly of 16- and 18-carbon chain lengths (14Trombe M.C. Laneelle M.A. Laneelle G. Biochim. Biophys. Acta. 1979; 574: 290-300Crossref PubMed Scopus (39) Google Scholar). 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 FabM, a trans-2, cis-3-decenoyl-ACP isomerase (15Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Enterococcus faecalis also has a fatty acid composition similar to E. coli, but also lacks a FabA, FabB, and FabM. However, E. faecalis has two FabZ homologs, and Wang and Cronan (16Wang H. Cronan J.E. J. Biol. Chem. 2004; 279: 34489-34495Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) show that one of these genes, now called FabN, functions as a dehydratase/isomerase analogous to FabA, whereas the other possessed only dehydratase activity (Fig. 1). Thus, one cannot predict the biochemical activity of this class of proteins based on bioinformatics. The comparison of the x-ray structures of FabA (10Leesong M. Henderson B.S. Gillig J.R. Schwab J.M. Smith J.L. Structure. 1996; 4: 253-264Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and FabZ (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), coupled with the analysis of site-directed mutants shows that the differences in the catalytic activities of the enzymes are not the result of distinct catalytic residues in the active site or the side chains of the residues that compose the substrate binding pocket (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Both proteins adopt a "hot dog" fold with a long central α-helix (the hot dog) surrounded by several β-sheets (the bun). A detailed comparison of these closely related structures led to the hypothesis that the different biochemistry of these enzymes is caused by differences in the β-sheet structures that alter the orientation of the central α-helix and thus the shape of the active site tunnel preventing the substrate from adopting a cis conformation in FabZ. The existence of two even more closely related enzymes, EfFabN and EfFabZ, with different catalytic properties but identical active site residues, allows us the opportunity to test the working hypothesis using domain swapping experiments to determine whether the isomerase activity is caused by the structure of the β-strands that control the shape of the active site tunnel. Materials—[2-14C]Malonyl-CoA (specific activity, 52 mCi/mmol) was from Amersham Biosciences; antibiotics, acyl-CoA, and E. coli ACP were from Sigma; molecular reagents, restriction enzymes, and T4 ligase were from Promega; pET vectors and expression strains were from Novagen; Ni2+-agarose resin was from Qiagen. Protein was quantitated by the Bradford method (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223639) Google Scholar). The Mycobacterium tuberculosis MtFabH, E. coli EcFabD, EcFabG, EcFabA, and S. pneumoniae SpFabF proteins were purified as described previously (12Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 18Choi K.-H. Kremer L. Besra G.S. Rock C.O. J. Biol. Chem. 2000; 275: 28201-28207Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 19Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 20Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 21Schujman G.E. Choi K.-H. Altabe S. Rock C.O. de Mendoza D. J. Bacteriol. 2001; 183: 3032-3040Crossref PubMed Scopus (66) Google Scholar). All other chemicals were reagent grade or better. Cloning and Construction of Chimeric Enzymes—The EffabN and EffabZ genes were amplified from genomic DNA of E. faecalis by primer sets EfFabNstart/EfFabNend and EfFabZstart/EfFabZend. The PCR products were ligated into plasmid pCR2.1 and sequenced. The plasmids were isolated and digested with NdeI and BamHI, and the gene fragments were isolated and ligated into plasmid pET15b digested with the same enzymes to generate the EfFabN and EfFabZ expression vectors pYL3 and pYL4. Chimeras were generated by overlapping PCR method. Sequences of internal primers are listed in Table I. T7 promoter primer with reverse primers and T7 terminator primer with forward primers were used to generate PCR fragments corresponding to the specific region of the two enzymes. After purification from agarose gels, these fragments were mixed with the right combination to serve as template, and T7 promoter and T7 terminator primers were used to amplify the recombinant molecule. PCR products from this step were ligated to PCR 2.1 vector and sequenced to ensure the chimeric constructs were correct. The chimeric genes were cut out with NdeI and BamHI and transferred into pET15b to generate the expression vectors.Table IPrimers used in this study All primers are listed in the 5′ → 3′ direction. Capital letters indicate complementarity to the sequence of EfFabN, and those in lower case indicate complementarity to the sequence of EfFabZ. NdeI and BamHI sites are underlined.PrimerSequenceEfFabNstartCGCCATATGAAAAAAGTAATGACTGCAACEfFabNendCGCGGATCCTTCTTATCGTCCCACAATEfFabZstartCGCCATATGAAATTAACAATTACAGAAATEfFabZendCGCGGATCCTTTTTCACCTATCCAATCAChr1revccaccaaagtatgccgtTTCACCTTCAAATTGATCCChr1forGGATCAATTTGAAGGTGAAacggcatactttggtggChr2revCCGCCAATATAGGCTGTtttccctttgaattcaggChr2forcctgaattcaaagggaaaACAGCCTATATTGGCGGChr3revATGTAATTTCAAGACATCgcctggtgttaccttttgChr3forcaaaaggtaacaccaggcGATGTCTTGAAATTACATChr4revGTCGCTTTGCCGATGCCagcagaagcgcggacttChr4foraagtccgcgcttctgctGGCATCGGCAAAGCGACChr5revctaaaattaatgtgtcACCAGGGACCACTTTTTGChr5forCAAAAAGTGGTCCCTGGTgacacattaattttagChr6revcacacctttacccattccGACAAAGTCACGTAATTChr6forATTACGTGACTTTGTCggaatgggtaaaggtgtgChr7revGTCGCTTTGCCGATGCCagcagaagcgcggacttChr7foraagtccgcgcttctgctGGCATCGGCAAAGCGAC Open table in a new tab Protein Purification—The pET15b plasmids were used to transform E. coli Rosetta-competent cells for protein expression. The selected transformants were cultured in LB medium with antibiotic (50 μg/ml carbenicillin and 34 μg/ml chloramphenicol) at 37 °C until A600 reached 0.6. Isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and incubation continued overnight at 20 °C. Cells were collected by centrifugation (6,000 rpm, 4 °C, 15 min), and cell pellets were lysed using a French press. Soluble proteins were applied to a Ni2+-agarose column and washed with 40 mm imidazole-containing metal chelation affinity chromatography buffer (20 mm Tris-HCl, pH 7.4, 0.5 m NaCl). His-tagged proteins were eluted with 500 mm imidazole in the same buffer. Proteins were quantitated by the Bradford method (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223639) Google Scholar). The purified proteins were stored at -20 °C. Relevant characteristics of the plasmids used in this study are shown in Table II.Table IIPlasmids used in this studyPlasmidRelevant characteristicspYL3pET15b carrying EffabNpYL4pET15b carrying EffabZpYL5pET15b carrying Chimera1pYL6pET15b carrying Chimera2pYL7pET15b carrying Chimera3pYL8pET15b carrying Chimera4pYL9pET15b carrying Chimera5pYL10pET15b carrying Chimera6pYL11pET15b carrying Chimera7 Open table in a new tab Purified EfFabN or EfFabZ proteins were applied to a Superdex 200 HR 16/60 column (Amersham Biosciences) and eluted with 20 mm Tris-HCl, pH 7.4, 1 mm EDTA, 100 mm NaCl, 1 mm dithiothreitol). The molecular masses of EfFabN and EfFabZ were estimated using globular protein standards and a calibration curve. Enzymatic Assays—The ability of the individual His6-tagged EfFabN and EfFabZ to carry out the dehydratase and/or isomerase activities was measured using a reconstituted system essentially as described previously (12Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 15Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Choi K.-H. Heath R.J. Rock C.O. J. Bacteriol. 2000; 182: 365-370Crossref PubMed Scopus (213) Google Scholar). The reaction mixtures contained 100 μm ACP, 1 mm β-mercaptoethanol, 0.1 m sodium phosphate buffer, pH 7.0, 100 μm NADPH, 50 μm octanoyl-CoA, 100 μm [2-14C]malonyl-CoA (specific activity, 52 mCi/mmol), 1.0 μg of MtFabH, 1.0 μg of EcFabD, 1.0 μg of EcFabG, 3 μg of SpFabF with 1.0 μg of EfFabN or 1.0 μg of EfFabZ or 1.0 μg of chimeric proteins in a final volume of 40 μl. The assay mixtures were incubated at 37 °C for 30 min and analyzed by conformationally sensitive gel electrophoresis in 13% polyacrylamide gels containing 2.5 m urea. Electrophoresis was performed at 25 °C and 32 mA/gel. The gels were dried, and the bands were quantitated using a PhosphorImager screen. In the experiments determining FabN isomerase activity, EcFabZ was added to the reaction mixture, and the reaction was incubated at 37 °C for 30 min before the addition of EfFabN. Specific activities were calculated from the slopes of the plot of product formation versus protein concentration in the assay. Characteristics of E. faecalis fab Genes—An analysis of the type II fatty acid biosynthetic genes in E. faecalis shows that they are located in two clusters in the genome (Fig. 2A). S. pneumoniae, a closely related bacteria that also synthesizes UFAs, has only one fab gene cluster in its genome corresponding to the large gene cluster in E. faecalis. A comparison of the predicted protein sequences of E. faecalis fab genes with that of S. pneumoniae showed that the 12-gene cluster contains all of the gene homologs of the S. pneumoniae fab gene cluster except fabM, which is an essential isomerase to make UFAs in S. pneumoniae (15Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 23Fozo E.M. Quivey R.G. J Bacteriol. 2004; 186: 4152-4158Crossref PubMed Scopus (99) Google Scholar). A separate 3-gene cluster contains homologs of E. coli fabI and fabF1, in addition to fabN, a fabZ homolog that possess the ability to introduce double bonds into growing acyl chains in vivo (16Wang H. Cronan J.E. J. Biol. Chem. 2004; 279: 34489-34495Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). FabT, the predicted transcriptional regulator in several Gram-positive bacterial fatty acid biosynthetic pathways (24Lu Y.-J. Zhang Y.-M. Rock C.O. Biochem. Cell Biol. 2004; 82: 145-155Crossref PubMed Scopus (127) Google Scholar), is located at the beginning of the 12-gene cluster. FabT belongs to the MarR superfamily, which are typically dimers that utilize a winged helix motif to bind a DNA palindrome (25Alekshun M.N. Kim Y.S. Levy S.B. Mol. Microbiol. 2000; 35: 1394-1404Crossref PubMed Scopus (80) Google Scholar, 26Alekshun M.N. Levy S.B. Mealy T.R. Seaton B.A. Head J.F. Nat. Struct. Biol. 2001; 8: 710-714Crossref PubMed Scopus (318) Google Scholar). Often bacterial transcription factors are autoregulated, and their DNA binding motifs are located within their own promoter regions. One DNA palindrome was found in the promoter region of FabT (Fig. 2A). Significantly, this same palindrome is found in the promoters of the fabI, fabF1, and fabK genes (Fig. 2A). Unraveling the transcriptional regulation in this large cluster is beyond the scope of this study. The significance of the bioinformatics analysis is that it ties the 3- and 12-gene clusters for fatty acid biosynthesis and suggests that the fabI, fabF1-fabN, and fabK genes may be coordinately regulated. Purification of EfFabN and EfFabZ—EfFabN and EfFabZ were cloned into pET15b vector and purified as described under "Experimental Procedures." Purified His-tagged EfFabZ and EfFabN had monomer molecular masses of ≈18 kDa based on SDS-gel electrophoresis, consistent with their primary sequence (Fig. 3). Recently, the crystal structure of Pseudomonas aeruginosa FabZ was solved, and it forms a classic "trimer of dimers" structure (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Accordingly, PaFabZ behaves as a hexamer exhibiting a Stokes radius on gel filtration chromatography corresponding to a 112-kDa protein (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), whereas EcFabA is a dimer both in its x-ray structure (10Leesong M. Henderson B.S. Gillig J.R. Schwab J.M. Smith J.L. Structure. 1996; 4: 253-264Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and in solution. Both EfFabN and EfFabZ are hexamers in solution as determined by gel filtration chromatography (Fig. 3), which illustrates that they have structures similar to that of PaFabZ. Enzyme Activity of EfFabN and EfFabZ—We used a complex, multicomponent reconstituted fatty acid biosynthetic system described under "Experimental Procedures" to detect the dehydratase and isomerase activities of purified EfFabN and EfFabZ in vitro (Fig. 4). This assay used enzymes from different sources to synthesize substrate for the dehydratases and followed the same principals as used previously in the characterization of the FabA and FabM isomerases (12Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 15Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The identify of the band indicated as β-hydroxy-cis-5-dodecenoyl-ACP was established by mass spectrometry (15Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The assay employed the MtFabH enzyme (from M. tuberculosis) to generate β-keto-[14C]decanoyl-ACP from octanoyl-CoA and [2-14C]malonyl-ACP (via EcFadD). The NADPH-dependent Ec-FabG reduced the intermediate to the substrate for the assays, β-hydroxy[14C]decanoyl-ACP (Fig. 4, lane 1). Addition of Sp-FabF, the elongation-condensing enzyme of S. pneumoniae, to the reaction did not, and should not, yield any new products (Fig. 4, lane 2). The addition of EcFabA (Fig. 4, lane 3) resulted in the conversion of the β-hydroxy intermediate to a mixture of trans-2- and cis-3-decenoyl-ACPs. The enoyl-ACP cannot be elongated by the SpFabF-condensing enzyme, but the cis-3 intermediate is a substrate. Accordingly, in the presence of SpFabF the cis-3-decenoyl-ACP is condensed with malonyl-ACP, and after reduction by EcFabG, a new band appeared on the gel corresponding to β-hydroxy-cis-5-dodecenoyl-ACP (Fig. 4, lane 4). Because this reaction mixture did not contain an enoyl-ACP reductase, additional rounds of elongation did not occur unless this product was isomerized again by FabA. FabA is selective for 10-carbon substrates and characteristically less active with unsaturated β-hydroxy intermediates (12Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 27795-27801Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), therefore there is only a trace conversion to β-hydroxy-14:2(Δ5,7)-ACP (the faint band below the β-hydroxy-12:1(Δ5)-ACP. The addition of EcFabZ converted the β-hydroxydecanoyl-ACP to the enoyl-ACP (Fig. 4, lane 5); however, the addition of SpFabF to this reaction did not lead to the appearance of the elongated 12-carbon unsaturated intermediate (Fig. 4, lane 6). The addition of EfFabN to the base reaction yielded enoyl-ACP (Fig. 4, lane 7), and β-hydroxy-cis-5-dodecanoyl-ACP appeared when SpFabF was added (Fig. 4, lane 8). Thus, EfFabN is capable of not only dehydrating β-hydroxydecanoyl-ACP but also isomerizing trans-2-decenoyl-ACP to cis-3-decenoyl-ACP in vitro. On the other hand, EfFabZ dehydrated β-hydroxydecanoyl-ACP (Fig. 4, lane 9) but lacked isomerase activity. These data establish that purified EfFabN is capable of isomerizing the trans-2-enoyl-ACP to the cis-3 intermediate, whereas EfFabZ cannot. This in vitro gel assay system was used to evaluate the relative activities of these two enzymes. The EcFabD/MtFabH/EcFabG system was used to present EfFabN and EfFabZ with β-hydroxydecanoyl-ACP as a dehydratase substrate. The formation of enoyl-ACP was measured as a function of EfFabN or EfFabZ concentration, and a specific activity calculated by linear regression curves (Fig. 5A). The EcFabD/MtFabH/Ec-FabG/EcFabZ system was used to provide the trans-2-enoyl-ACP substrate for measuring isomerase activity. The formation of an elongation product with an excess concentration of Sp-FabF arising from EfFabN isomerase activity as a function of EfFabN in the assay was used to determine the isomerase-specific activity of EfFabN (Fig. 5B). The dehydratase activity of EfFabN, 0.07 ± 0.002 pmol/min/ng, was much less than that of EfFabZ, which was 2.2 ± 0.07 pmol/min/ng, whereas the dehydratase activities of the two controls, EcFabA and EcFabZ, were 0.34 ± 0.03 and 0.36 ± 0.003 pmol/min/ng, respectively. Thus, EfFabN had the lowest dehydratase activity of the four enzymes in the coupled assay. The isomerase activity of Ef-FabN for the 10-carbon substrate was 0.6 ± 0.03 pmol/min/μg, whereas the specific activity of EcFabA under these same assay conditions was 52 ± 1 pmol/min/μg (Fig. 5B). Thus, FabN was 86-fold less efficient than EcFabA in the formation of cis-double bonds in the in vitro fatty acid synthase assay reconstituted with the indicated constellation of E. coli enzymes and ACP cofactor. Also, FabN was about 2-fold less active than the FabM isomerase required for unsaturated fatty acid synthesis in S. pneumoniae (15Marrakchi H. Choi K.-H. Rock C.O. J. Biol. Chem. 2002; 277: 44809-44816Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Although this assay is capable of clearly distinguishing dehydratases from isomerases, the activities of the FabM and FabN isomerases are low compared with FabA. The reasons for the low activities of the Gram-positive isomerases in this reconstituted assay are attributed in part to the use of heterologous ACP and elongation-condensing enzymes in the experiment. These data are consistent with the inability of fabN expression to complement the growth defect in fabA(Ts) mutants (16Wang H. Cronan J.E. J. Biol. Chem. 2004; 279: 34489-34495Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Chimera Construction and Enzyme Activity—The working hypothesis developed from our structural analysis of EcFabA and PaFabZ (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) was that the isomerase activity depends on the shape of the substrate binding tunnel, which is controlled by the positioning of the β-strands (the bun) surrounding the long central helix (the hot dog). The existence of these two closely related FabZ enzymes with different catalytic properties provided an opportunity to test this hypothesis concerning the basis for their distinct catalytic properties. The goal was to determine the minimal structural features required to convert a dehydratase (EfFabZ) into a dehydratase/isomerase (Ef-FabN). Structural models of EfFabN and EfFabZ were generated using the PaFabZ x-ray structure (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) as the template. There are several amino acid differences between EfFabN and EfFabZ which were predicted to reside in the substrate binding tunnel and which might be responsible for the isomerase activity of EfFabN. These include Asn-16, Ile-20, Thr-57, Ile-88, and Asn-92. We introduced each of these point mutations into EcFabZ and measured their activities. All of the mutants retained dehydratase activity, and none acquired the isomerase function (data not shown). The next approach was to construct a series of chimeric EfFabZ/N proteins to determine the minimum structural elements required to transform EfFabZ into an isomerase. The transition points for chimera construction were selected by based upon the structure of PaFabZ (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and are mapped onto the primary sequences in Fig. 2B. EfFabN and EfFabZ both have high similarity to each other and PaFabZ (EfFabN has 55% similarity and 41.6% identity to PaFabZ; EfFabZ has 56.5% similarity and 43.5% identity to PaFabZ), and their structural folding patterns are clearly predicted to be similar to those determined for PaFabZ. The divisions between chimeras were made at the boundary of loops between the β-sheet structures with at least one identical residue in both proteins occurring after the joint (Fig. 2B). Fig. 2C depicts schematically the chimeras made from introducing different EfFabN domains into EfFabZ. All chimeras were hexamers as judged by gel filtration chromatography and retained catalytic activity. First, EfFabN and Ef-FabZ were divided to two parts, and a swap of the corresponding regions of EfFabN and EfFabZ was made to generate Chimera1 and Chimera2. The analysis of these two proteins showed that both of them retained the ability to dehydrate β-hydroxydecanoyl-ACP to enoyl ACP (Fig. 6, lanes 7 and 8). On the other hand, only Chimera2 containing the C-terminal part of EfFabN possessed 43% of the isomerase activity of EfFabN as indicated by the formation of the β-hydroxy-cis-5-dodecenoyl-ACP (Fig. 6, lane 8). We then divided the C-terminal part of EfFabN into three pieces to narrow down the region. Chimera3 and Chimera4 had only dehydratase activity (Fig. 6, lanes 9 and 10), leading to the conclusion that β3 and the following loop region of EfFabN were essential requirements for isomerase activity. However, the fact that Chimera5 lacked isomerase activity (Fig. 6, lane 11) illustrated that β3 and following loop were not sufficient to support isomerase activity. The combination of β3 and β4 (Chimera6) possessed isomerase activity at 38% of the Ef-FabN level (Fig. 6, lane 12), whereas the combination of β3 with β5 and β6 (Chimera7) did not (Fig. 6, lane 13). These data show that the β3–β4 region of EfFabN was necessary and sufficient to transform EfFabZ from a dehydratase to a dehydratase/isomerase. The isomerase activity of Chimera6 showed the important role of the combination of the β3/β4 strands in specifying isomerase activity. Two residues, Phe-86 from β3 and Val-108 from β4 in EfFabZ, were candidates for mediating this effect based on the modeling of EfFabN and EfFabZ using the structure of PaFabZ as a template. The corresponding residues in EfFabN are Ile-88 and Phe-110. In both structures, these two residues sit face to face on the outside of the α3-helix in a position to modulate the orientation of the helix, therefore, we prepared single and double mutants to replace these residues and assessed the isomerase activity of the constructs. EfFabZ(F86I), EfFabZ(V108F), and EfFabZ(F86I, V108F) were all found to retain dehydratase activity, but all three mutants lacked the ability to isomerize the substrate (not shown). These data suggest that the conversion of EfFabZ to EfFabN requires the sum of the differences between the two β3/β4 strands, rather than a simple single amino acid substitution. Conclusions—These data are consistent with the hypothesis that the shape of the substrate binding tunnel is the major determinant of the isomerase activity of the FabZ class of β-hydroxyacyl-ACP dehydratases. The replacement of the β3/β4 strands in EfFabZ with the corresponding strands from EfFabN were the minimal requirements to convert EfFabZ from a dehydratase to a dehydratase/isomerase. Molecular modeling of the EfFabZ and EfFabN clearly indicate that their structures are both very similar to the PaFabZ crystal structure. The β3/β4 strands that control the catalytic activity of the enzymes interact with both ends of the central helix α3, and the conformation of these strands along this interaction interface determines the orientation of helix α3, and hence the shape of the active site tunnel (Fig. 7). Single and double mutants within these strands failed to transform EfFabZ into an isomerase, only when the β3/β4 strands were inserted as a unit was isomerase activity reconstituted. The structural analysis of FabA and FabZ (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) led to the hypothesis that differences in the shapes of the active site tunnels caused by the structures of these β strands, rather than active site chemistry, explain why the two enzymes differ in their ability to carry out the isomerization reaction. EcFabA (10Leesong M. Henderson B.S. Gillig J.R. Schwab J.M. Smith J.L. Structure. 1996; 4: 253-264Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) and PaFabZ (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) possess long, narrow, hydrophobic tunnels that span both monomers. The substrate tunnels both begin at the surface with narrow openings that can be completely occluded by a tyrosine, snake under the N terminus of the long, central α3-helix of the first monomer, and extend along the side of the corresponding helix α3 from the second monomer on its way back to the surface (Fig. 7). The tunnels are roughly 20 Å long, and the catalytic residues are located about halfway down their length. Both proteins have an active site histidine that acts as a general base with the Nδ atom hydrogen-bonded to a backbone carbonyl oxygen. Both proteins also have catalytic water molecules that are held in place by hydrogen bonds to a backbone amide at the N-terminal end of the central helix and to an acidic residue, Asp-84 in EcFabA and Glu-68 in EcFabZ. Although the acidic residues are different, they hold the water molecules in essentially the same spatial position within the tunnels. Site-directed mutagenesis swapping the aspartate in FabA for the glutamate in FabZ, and vice versa, do not change the catalytic properties of the proteins, making it clear that the catalytic machinery is equivalent and capable of carrying out both the dehydratase and isomerase activities (11Kimber M.S. Martin F. Lu Y. Houston S. Vedadi M. Dharamsi A. Feibig K.M. Schmid M. Rock C.O. J. Biol. Chem. 2004; 279: 52593-52602Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Rather, the ability of FabA, but not FabZ, to place the trans-2 substrate in an appropriate conformation to allow isomerization was proposed to arise from the differences in the protein backbones in the distal part of the substrate binding tunnels, encompassing helix α3 and strands β3/β4. Differences in the orientation of helix α3 are the primary structural differences leading to differently shaped active site tunnels (Fig. 7), which in turn position on the substrate in a conformation that in FabA/N, but not FabZ, is appropriate for the isomerization reaction. Strand β4 pushes the α3-helix upward, and strand β3 moves in the same direction to allow this movement and form one side of the active site tunnel (Fig. 7). Thus, the catalytic properties of the FabA/Z class of dehydratases is specified by the β3/β4 strands, not the catalytic residues, which alter the shapes of the active site tunnels.
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