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• W2022719417 abstract "The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACPPks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACPPks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening. The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACPPks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACPPks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening. Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1.Marrakchi H. Bardou F. Lanéelle M.A. Daffé M. Daffé M. Reyrat J.-M. The Mycobacterial Cell Envelope. ASM Press, Washington, DC2008: 41-62Google Scholar). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2.Zuber B. Chami M. Houssin C. Dubochet J. Griffiths G. Daffé M. J. Bacteriol. 2008; 190: 5672-5680Crossref PubMed Scopus (326) Google Scholar, 3.Hoffmann C. Leis A. Niederweis M. Plitzko J.M. Engelhardt H. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3963-3967Crossref PubMed Scopus (433) Google Scholar) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4.Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar).The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5.Walker R.W. Prome J.C. Lacave C.S. Biochim. Biophys. Acta. 1973; 326: 52-62Crossref PubMed Scopus (49) Google Scholar, 6.Shimakata T. Iwaki M. Kusaka T. Arch. Biochem. Biophys. 1984; 229: 329-339Crossref PubMed Scopus (25) Google Scholar) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1.Marrakchi H. Bardou F. Lanéelle M.A. Daffé M. Daffé M. Reyrat J.-M. The Mycobacterial Cell Envelope. ASM Press, Washington, DC2008: 41-62Google Scholar), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7.Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar, 8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 9.Gande R. Gibson K.J. Brown A.K. Krumbach K. Dover L.G. Sahm H. Shioyama S. Oikawa T. Besra G.S. Eggeling L. J. Biol. Chem. 2004; 279: 44847-44857Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC) 3The abbreviations used are: ACCacyl-CoA carboxylaseACPacyl carrier proteinATacyl transferaseC-ACPC-terminal ACP domainKSketosynthaseLC-ESI-MS/MSliquid chromatography-electrospray ionization-tandem mass spectrometryN-ACPN-terminal ACP domainNRPSnonribosomal peptide synthaseNRPS-PKShybrid NRPS-PKS systemPKSpolyketide synthaseP-pant4′-phosphopantetheinylTLCthin-layer chromatographyHPLChigh pressure liquid chromatographyWTwild typeGC-MSgas chromatography-mass spectrometry.3The abbreviations used are: ACCacyl-CoA carboxylaseACPacyl carrier proteinATacyl transferaseC-ACPC-terminal ACP domainKSketosynthaseLC-ESI-MS/MSliquid chromatography-electrospray ionization-tandem mass spectrometryN-ACPN-terminal ACP domainNRPSnonribosomal peptide synthaseNRPS-PKShybrid NRPS-PKS systemPKSpolyketide synthaseP-pant4′-phosphopantetheinylTLCthin-layer chromatographyHPLChigh pressure liquid chromatographyWTwild typeGC-MSgas chromatography-mass spectrometry. complex that most likely activates, through a C2-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 10.Gande R. Dover L.G. Krumbach K. Besra G.S. Sahm H. Oikawa T. Eggeling L. J. Bacteriol. 2007; 189: 5257-5264Crossref PubMed Scopus (79) Google Scholar), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12.Trivedi O.A. Arora P. Sridharan V. Tickoo R. Mohanty D. Gokhale R.S. Nature. 2004; 428: 441-445Crossref PubMed Scopus (223) Google Scholar), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13.Lea-Smith D.J. Pyke J.S. Tull D. McConville M.J. Coppel R.L. Crellin P.K. J. Biol. Chem. 2007; 282: 11000-11008Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (see Fig. 1).Although the enzymatic properties of the ACC complex have been well characterized (9.Gande R. Gibson K.J. Brown A.K. Krumbach K. Dover L.G. Sahm H. Shioyama S. Oikawa T. Besra G.S. Eggeling L. J. Biol. Chem. 2004; 279: 44847-44857Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14.Chalut C. Botella L. de Sousa-D'Auria C. Houssin C. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8511-8516Crossref PubMed Scopus (63) Google Scholar). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids.DiscussionThe present work demonstrated that the activated Pks13 enzyme of M. tuberculosis, in adequate experimental conditions, has a condensing activity in vitro and is able to synthesize, in coupled reaction with FadD32, the biosynthetic precursors of mycolic acids, α-alkyl β-ketoacids, from a fatty acyl-AMP and a 2-carboxyacyl-CoA (Fig. 8). For a matter of both solubility and availability of radiolabeled molecules, the function of Pks13 was studied in the presence of substrates shorter than its presumed natural substrates (C24-C26 carboxyacyl-CoAs and C40-C60 meromycoloyl-AMP) within mycobacteria. Nevertheless, the fact that Pks13 of M. tuberculosis is able to condense relatively short chain substrates (C12, C16), equivalent to those used by the condensing enzyme from Corynebacterium, correlates with the production of C32-C34 corynomycolates upon heterologous complementation of a C. glutamicum pks13 mutant strain by the M. tuberculosis pks13 gene. 4C. de Sousa d'Auria and C. Houssin, personal communication. If the Pks13 enzyme from M. tuberculosis presents a large specificity toward the chain length of its substrates, the length of the mero and branch chains of mycolic acids might be controlled by the enzymes that activate Pks13 substrates, i.e. FadD32 and the acyl-CoA carboxylase complex AccA3-AccD4 (Fig. 1). Consistently, it has been recently shown that AccA3-AccD4 from M. tuberculosis exhibits no activity in the presence acyl-CoA shorter than C24-C26, which perfectly matches with the required size of the extender unit during mycolic condensation in this bacterial species (Fig. 1) (11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).FIGURE 8Scheme of the stepwise activity of FadD32-Pks13 PKS and its domain organization. To simplify, C16 acyl chains were drawn. FadD32 synthesizes meromycoloyl-AMPs from the meromycolic acids and ATP (1). The meromycoloyl chain of these intermediates is then specifically loaded by FadD32 onto the P-pant arm of the N-ACP domain of Pks13 (2). This is an acyl-AMP/ACP transacylation. The meromycoloyl chain is then transferred onto the KS domain (3). The extender unit carboxyacyl-CoA is specifically loaded onto the AT domain, which catalyzes the covalent attachment of the carboxyacyl chain to its active site (1′) and its subsequent transfer specifically onto the C-ACP domain (2′). The KS domain catalyzes the Claisen-type condensation between the meromycoloyl and the carboxyacyl chains to produce a α-alkyl β-ketothioester linked to the C-ACP domain (3′). Then, it is likely that the thioesterase domain catalyzes the release of the α-alkyl β-ketoacyl chain and its transfer onto an unknown acceptor (X1) (4′). Thus, FadD32 constitutes one of the two domains of the loading module of the polyketide synthase formed by both FadD32 and Pks13 polypeptides.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Investigation of the different steps involved in condensation resulted in the determination of the general catalytic scheme leading to the formation of the α-alkyl β-ketoacids (Fig. 8). The carboxyacyl-CoA unit is loaded onto Pks13 via its AT domain. The latter has a predilection for both long chain and carboxylated molecules. Pks13 is an unprecedented polyketide synthase because its extender units do not correspond to the short classical units, malonyl-CoA or methyl-malonyl-CoA, used by the other PKSs. The predilection of this enzyme for carboxyacyl-CoAs of unusual chain length is reflected in the primary structure of its AT domain. Indeed, the latter is relatively distant from the primary structure of the AT domain of other PKSs and fatty acid synthases, as displayed by the distance tree obtained by Blast alignment (supplementary Fig. S6).Our data showed that the acyl chain of the acyl-AMPs produced by the FadD32 enzyme in the presence of a fatty acid and ATP are specifically transferred onto the N-ACP domain of Pks13 (Fig. 8). There are two possible mechanisms of acylation in trans: (i) release of the acyl-AMPs by FadD32 and adventitious acylation of the reactive –SH group of the P-pant arm of N-ACPPks13 and (ii) enzyme-dependent transfer of the acyl chain from AMP to Pks13. The present work demonstrated that the transfer cannot be undertaken by the AT domain of Pks13 in vitro, but is dependent upon the presence of active FadD32 that catalyzes a reaction of acyl-AMP/N-ACPPks13 transacylation (Fig. 8). Moreover, using Pks13 mutant proteins, we showed that FadD32 is unable to load an acyl chain onto the C-ACPPks13 domain. The selectivity of FadD32 for the N-ACP domain of Pks13 might be facilitated by the substantial sequence divergence (24% identity) between the two ACP-like domains of Pks13. The specificity between an adenylation enzyme and an ACP protein has been described for PKS and NRPS (24.Revill W.P. Bibb M.J. Hopwood D.A. J. Bacteriol. 1996; 178: 5660-5667Crossref PubMed Google Scholar, 25.Schmoock G. Pfennig F. Jewiarz J. Schlumbohm W. Laubinger W. Schauwecker F. Keller U. J. Biol. Chem. 2005; 280: 4339-4349Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, to the best of our knowledge, this is the first demonstration of an enzyme-dependent loading mechanism in the case of PKSs. Interestingly, we have observed that FadD32 could not be replaced by FadD26, a mycobacterial FAAL, during the condensation reaction, although FadD26 was able to load the acyl chain of its acyl-AMP products onto Pks13 in vitro. A privileged interaction between FadD32 and Pks13 might be required for proper folding of the PKS necessary to the subsequent steps of transfer and catalysis. The fact that the fadD32 gene is essential in mycobacteria (8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) is consistent with our data and proves that none of the FadD enzymes display a redundant activity in vivo. One can reasonably propose that, in vivo, FadD32 activates the very long meromycolic acids into meromycoloyl-AMPs and transfers the meromycolic acyl chains onto N-ACPPks13. This is reminiscent of the double function of adenylation and transfer described for some so-called “adenylation domains” found in NRPS or NRPS-PKS enzymes (26.Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar). As in the case of these enzymes, one can propose that FadD32 together with N-ACPPks13 correspond to the “loading module” of a PKS formed by both FadD32 and Pks13 (Fig. 8). The discrete FadD32 enzyme is reminiscent of the lone standing salicyl-AMP ligase domains MbtA and YbtE found in the hybrid NRPS-PKSs involved in mycobactin and yersiniabactin biosyntheses, respectively (26.Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 27.Gehring A.M. Mori I. Perry R.D. Walsh C.T. Biochemistry. 1998; 37: 11637-11650Crossref PubMed Scopus (125) Google Scholar). The knowledge of Pks13 enzymatic properties as well as of the experimental conditions for in vitro activity assays will now serve as fundamental tools for screening for inhibitors of this very original condensing enzyme that, because of its essentiality in mycobacteria (7.Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar) and its characteristic features, represents an excellent target for novel antimycobacterial drug design. Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1.Marrakchi H. Bardou F. Lanéelle M.A. Daffé M. Daffé M. Reyrat J.-M. The Mycobacterial Cell Envelope. ASM Press, Washington, DC2008: 41-62Google Scholar). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2.Zuber B. Chami M. Houssin C. Dubochet J. Griffiths G. Daffé M. J. Bacteriol. 2008; 190: 5672-5680Crossref PubMed Scopus (326) Google Scholar, 3.Hoffmann C. Leis A. Niederweis M. Plitzko J.M. Engelhardt H. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3963-3967Crossref PubMed Scopus (433) Google Scholar) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4.Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5.Walker R.W. Prome J.C. Lacave C.S. Biochim. Biophys. Acta. 1973; 326: 52-62Crossref PubMed Scopus (49) Google Scholar, 6.Shimakata T. Iwaki M. Kusaka T. Arch. Biochem. Biophys. 1984; 229: 329-339Crossref PubMed Scopus (25) Google Scholar) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1.Marrakchi H. Bardou F. Lanéelle M.A. Daffé M. Daffé M. Reyrat J.-M. The Mycobacterial Cell Envelope. ASM Press, Washington, DC2008: 41-62Google Scholar), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7.Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar, 8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 9.Gande R. Gibson K.J. Brown A.K. Krumbach K. Dover L.G. Sahm H. Shioyama S. Oikawa T. Besra G.S. Eggeling L. J. Biol. Chem. 2004; 279: 44847-44857Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC) 3The abbreviations used are: ACCacyl-CoA carboxylaseACPacyl carrier proteinATacyl transferaseC-ACPC-terminal ACP domainKSketosynthaseLC-ESI-MS/MSliquid chromatography-electrospray ionization-tandem mass spectrometryN-ACPN-terminal ACP domainNRPSnonribosomal peptide synthaseNRPS-PKShybrid NRPS-PKS systemPKSpolyketide synthaseP-pant4′-phosphopantetheinylTLCthin-layer chromatographyHPLChigh pressure liquid chromatographyWTwild typeGC-MSgas chromatography-mass spectrometry.3The abbreviations used are: ACCacyl-CoA carboxylaseACPacyl carrier proteinATacyl transferaseC-ACPC-terminal ACP domainKSketosynthaseLC-ESI-MS/MSliquid chromatography-electrospray ionization-tandem mass spectrometryN-ACPN-terminal ACP domainNRPSnonribosomal peptide synthaseNRPS-PKShybrid NRPS-PKS systemPKSpolyketide synthaseP-pant4′-phosphopantetheinylTLCthin-layer chromatographyHPLChigh pressure liquid chromatographyWTwild typeGC-MSgas chromatography-mass spectrometry. complex that most likely activates, through a C2-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 10.Gande R. Dover L.G. Krumbach K. Besra G.S. Sahm H. Oikawa T. Eggeling L. J. Bacteriol. 2007; 189: 5257-5264Crossref PubMed Scopus (79) Google Scholar), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12.Trivedi O.A. Arora P. Sridharan V. Tickoo R. Mohanty D. Gokhale R.S. Nature. 2004; 428: 441-445Crossref PubMed Scopus (223) Google Scholar), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13.Lea-Smith D.J. Pyke J.S. Tull D. McConville M.J. Coppel R.L. Crellin P.K. J. Biol. Chem. 2007; 282: 11000-11008Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (see Fig. 1). acyl-CoA carboxylase acyl carrier protein acyl transferase C-terminal ACP domain ketosynthase liquid chromatography-electrospray ionization-tandem mass spectrometry N-terminal ACP domain nonribosomal peptide synthase hybrid NRPS-PKS system polyketide synthase 4′-phosphopantetheinyl thin-layer chromatography high pressure liquid chromatography wild type gas chromatography-mass spectrometry. acyl-CoA carboxylase acyl carrier protein acyl transferase C-terminal ACP domain ketosynthase liquid chromatography-electrospray ionization-tandem mass spectrometry N-terminal ACP domain nonribosomal peptide synthase hybrid NRPS-PKS system polyketide synthase 4′-phosphopantetheinyl thin-layer chromatography high pressure liquid chromatography wild type gas chromatography-mass spectrometry. Although the enzymatic properties of the ACC complex have been well characterized (9.Gande R. Gibson K.J. Brown A.K. Krumbach K. Dover L.G. Sahm H. Shioyama S. Oikawa T. Besra G.S. Eggeling L. J. Biol. Chem. 2004; 279: 44847-44857Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14.Chalut C. Botella L. de Sousa-D'Auria C. Houssin C. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 8511-8516Crossref PubMed Scopus (63) Google Scholar). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids. DiscussionThe present work demonstrated that the activated Pks13 enzyme of M. tuberculosis, in adequate experimental conditions, has a condensing activity in vitro and is able to synthesize, in coupled reaction with FadD32, the biosynthetic precursors of mycolic acids, α-alkyl β-ketoacids, from a fatty acyl-AMP and a 2-carboxyacyl-CoA (Fig. 8). For a matter of both solubility and availability of radiolabeled molecules, the function of Pks13 was studied in the presence of substrates shorter than its presumed natural substrates (C24-C26 carboxyacyl-CoAs and C40-C60 meromycoloyl-AMP) within mycobacteria. Nevertheless, the fact that Pks13 of M. tuberculosis is able to condense relatively short chain substrates (C12, C16), equivalent to those used by the condensing enzyme from Corynebacterium, correlates with the production of C32-C34 corynomycolates upon heterologous complementation of a C. glutamicum pks13 mutant strain by the M. tuberculosis pks13 gene. 4C. de Sousa d'Auria and C. Houssin, personal communication. If the Pks13 enzyme from M. tuberculosis presents a large specificity toward the chain length of its substrates, the length of the mero and branch chains of mycolic acids might be controlled by the enzymes that activate Pks13 substrates, i.e. FadD32 and the acyl-CoA carboxylase complex AccA3-AccD4 (Fig. 1). Consistently, it has been recently shown that AccA3-AccD4 from M. tuberculosis exhibits no activity in the presence acyl-CoA shorter than C24-C26, which perfectly matches with the required size of the extender unit during mycolic condensation in this bacterial species (Fig. 1) (11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Investigation of the different steps involved in condensation resulted in the determination of the general catalytic scheme leading to the formation of the α-alkyl β-ketoacids (Fig. 8). The carboxyacyl-CoA unit is loaded onto Pks13 via its AT domain. The latter has a predilection for both long chain and carboxylated molecules. Pks13 is an unprecedented polyketide synthase because its extender units do not correspond to the short classical units, malonyl-CoA or methyl-malonyl-CoA, used by the other PKSs. The predilection of this enzyme for carboxyacyl-CoAs of unusual chain length is reflected in the primary structure of its AT domain. Indeed, the latter is relatively distant from the primary structure of the AT domain of other PKSs and fatty acid synthases, as displayed by the distance tree obtained by Blast alignment (supplementary Fig. S6).Our data showed that the acyl chain of the acyl-AMPs produced by the FadD32 enzyme in the presence of a fatty acid and ATP are specifically transferred onto the N-ACP domain of Pks13 (Fig. 8). There are two possible mechanisms of acylation in trans: (i) release of the acyl-AMPs by FadD32 and adventitious acylation of the reactive –SH group of the P-pant arm of N-ACPPks13 and (ii) enzyme-dependent transfer of the acyl chain from AMP to Pks13. The present work demonstrated that the transfer cannot be undertaken by the AT domain of Pks13 in vitro, but is dependent upon the presence of active FadD32 that catalyzes a reaction of acyl-AMP/N-ACPPks13 transacylation (Fig. 8). Moreover, using Pks13 mutant proteins, we showed that FadD32 is unable to load an acyl chain onto the C-ACPPks13 domain. The selectivity of FadD32 for the N-ACP domain of Pks13 might be facilitated by the substantial sequence divergence (24% identity) between the two ACP-like domains of Pks13. The specificity between an adenylation enzyme and an ACP protein has been described for PKS and NRPS (24.Revill W.P. Bibb M.J. Hopwood D.A. J. Bacteriol. 1996; 178: 5660-5667Crossref PubMed Google Scholar, 25.Schmoock G. Pfennig F. Jewiarz J. Schlumbohm W. Laubinger W. Schauwecker F. Keller U. J. Biol. Chem. 2005; 280: 4339-4349Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, to the best of our knowledge, this is the first demonstration of an enzyme-dependent loading mechanism in the case of PKSs. Interestingly, we have observed that FadD32 could not be replaced by FadD26, a mycobacterial FAAL, during the condensation reaction, although FadD26 was able to load the acyl chain of its acyl-AMP products onto Pks13 in vitro. A privileged interaction between FadD32 and Pks13 might be required for proper folding of the PKS necessary to the subsequent steps of transfer and catalysis. The fact that the fadD32 gene is essential in mycobacteria (8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) is consistent with our data and proves that none of the FadD enzymes display a redundant activity in vivo. One can reasonably propose that, in vivo, FadD32 activates the very long meromycolic acids into meromycoloyl-AMPs and transfers the meromycolic acyl chains onto N-ACPPks13. This is reminiscent of the double function of adenylation and transfer described for some so-called “adenylation domains” found in NRPS or NRPS-PKS enzymes (26.Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar). As in the case of these enzymes, one can propose that FadD32 together with N-ACPPks13 correspond to the “loading module” of a PKS formed by both FadD32 and Pks13 (Fig. 8). The discrete FadD32 enzyme is reminiscent of the lone standing salicyl-AMP ligase domains MbtA and YbtE found in the hybrid NRPS-PKSs involved in mycobactin and yersiniabactin biosyntheses, respectively (26.Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 27.Gehring A.M. Mori I. Perry R.D. Walsh C.T. Biochemistry. 1998; 37: 11637-11650Crossref PubMed Scopus (125) Google Scholar). The knowledge of Pks13 enzymatic properties as well as of the experimental conditions for in vitro activity assays will now serve as fundamental tools for screening for inhibitors of this very original condensing enzyme that, because of its essentiality in mycobacteria (7.Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar) and its characteristic features, represents an excellent target for novel antimycobacterial drug design. The present work demonstrated that the activated Pks13 enzyme of M. tuberculosis, in adequate experimental conditions, has a condensing activity in vitro and is able to synthesize, in coupled reaction with FadD32, the biosynthetic precursors of mycolic acids, α-alkyl β-ketoacids, from a fatty acyl-AMP and a 2-carboxyacyl-CoA (Fig. 8). For a matter of both solubility and availability of radiolabeled molecules, the function of Pks13 was studied in the presence of substrates shorter than its presumed natural substrates (C24-C26 carboxyacyl-CoAs and C40-C60 meromycoloyl-AMP) within mycobacteria. Nevertheless, the fact that Pks13 of M. tuberculosis is able to condense relatively short chain substrates (C12, C16), equivalent to those used by the condensing enzyme from Corynebacterium, correlates with the production of C32-C34 corynomycolates upon heterologous complementation of a C. glutamicum pks13 mutant strain by the M. tuberculosis pks13 gene. 4C. de Sousa d'Auria and C. Houssin, personal communication. If the Pks13 enzyme from M. tuberculosis presents a large specificity toward the chain length of its substrates, the length of the mero and branch chains of mycolic acids might be controlled by the enzymes that activate Pks13 substrates, i.e. FadD32 and the acyl-CoA carboxylase complex AccA3-AccD4 (Fig. 1). Consistently, it has been recently shown that AccA3-AccD4 from M. tuberculosis exhibits no activity in the presence acyl-CoA shorter than C24-C26, which perfectly matches with the required size of the extender unit during mycolic condensation in this bacterial species (Fig. 1) (11.Oh T.J. Daniel J. Kim H.J. Sirakova T.D. Kolattukudy P.E. J. Biol. Chem. 2006; 281: 3899-3908Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Investigation of the different steps involved in condensation resulted in the determination of the general catalytic scheme leading to the formation of the α-alkyl β-ketoacids (Fig. 8). The carboxyacyl-CoA unit is loaded onto Pks13 via its AT domain. The latter has a predilection for both long chain and carboxylated molecules. Pks13 is an unprecedented polyketide synthase because its extender units do not correspond to the short classical units, malonyl-CoA or methyl-malonyl-CoA, used by the other PKSs. The predilection of this enzyme for carboxyacyl-CoAs of unusual chain length is reflected in the primary structure of its AT domain. Indeed, the latter is relatively distant from the primary structure of the AT domain of other PKSs and fatty acid synthases, as displayed by the distance tree obtained by Blast alignment (supplementary Fig. S6). Our data showed that the acyl chain of the acyl-AMPs produced by the FadD32 enzyme in the presence of a fatty acid and ATP are specifically transferred onto the N-ACP domain of Pks13 (Fig. 8). There are two possible mechanisms of acylation in trans: (i) release of the acyl-AMPs by FadD32 and adventitious acylation of the reactive –SH group of the P-pant arm of N-ACPPks13 and (ii) enzyme-dependent transfer of the acyl chain from AMP to Pks13. The present work demonstrated that the transfer cannot be undertaken by the AT domain of Pks13 in vitro, but is dependent upon the presence of active FadD32 that catalyzes a reaction of acyl-AMP/N-ACPPks13 transacylation (Fig. 8). Moreover, using Pks13 mutant proteins, we showed that FadD32 is unable to load an acyl chain onto the C-ACPPks13 domain. The selectivity of FadD32 for the N-ACP domain of Pks13 might be facilitated by the substantial sequence divergence (24% identity) between the two ACP-like domains of Pks13. The specificity between an adenylation enzyme and an ACP protein has been described for PKS and NRPS (24.Revill W.P. Bibb M.J. Hopwood D.A. J. Bacteriol. 1996; 178: 5660-5667Crossref PubMed Google Scholar, 25.Schmoock G. Pfennig F. Jewiarz J. Schlumbohm W. Laubinger W. Schauwecker F. Keller U. J. Biol. Chem. 2005; 280: 4339-4349Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, to the best of our knowledge, this is the first demonstration of an enzyme-dependent loading mechanism in the case of PKSs. Interestingly, we have observed that FadD32 could not be replaced by FadD26, a mycobacterial FAAL, during the condensation reaction, although FadD26 was able to load the acyl chain of its acyl-AMP products onto Pks13 in vitro. A privileged interaction between FadD32 and Pks13 might be required for proper folding of the PKS necessary to the subsequent steps of transfer and catalysis. The fact that the fadD32 gene is essential in mycobacteria (8.Portevin D. de Sousa-D'Auria C. Montrozier H. Houssin C. Stella A. Lanéelle M.A. Bardou F. Guilhot C. Daffé M. J. Biol. Chem. 2005; 280: 8862-8874Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) is consistent with our data and proves that none of the FadD enzymes display a redundant activity in vivo. One can reasonably propose that, in vivo, FadD32 activates the very long meromycolic acids into meromycoloyl-AMPs and transfers the meromycolic acyl chains onto N-ACPPks13. This is reminiscent of the double function of adenylation and transfer described for some so-called “adenylation domains” found in NRPS or NRPS-PKS enzymes (26.Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar). As in the case of these enzymes, one can propose that FadD32 together with N-ACPPks13 correspond to the “loading module” of a PKS formed by both FadD32 and Pks13 (Fig. 8). The discrete FadD32 enzyme is reminiscent of the lone standing salicyl-AMP ligase domains MbtA and YbtE found in the hybrid NRPS-PKSs involved in mycobactin and yersiniabactin biosyntheses, respectively (26.Quadri L.E. Sello J. Keating T.A. Weinreb P.H. Walsh C.T. Chem. Biol. 1998; 5: 631-645Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 27.Gehring A.M. Mori I. Perry R.D. Walsh C.T. Biochemistry. 1998; 37: 11637-11650Crossref PubMed Scopus (125) Google Scholar). The knowledge of Pks13 enzymatic properties as well as of the experimental conditions for in vitro activity assays will now serve as fundamental tools for screening for inhibitors of this very original condensing enzyme that, because of its essentiality in mycobacteria (7.Portevin D. De Sousa-D'Auria C. Houssin C. Grimaldi C. Chami M. Daffé M. Guilhot C. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 314-319Crossref PubMed Scopus (276) Google Scholar) and its characteristic features, represents an excellent target for novel antimycobacterial drug design. We are grateful to F. Laval and A. Lemassu (IPBS) for technical help, C. Bon, N. Eynard, V. Guillet, M.-A. Lanéelle, W. Malaga, and P. Roblin (IPBS) for precious advice, and C. Guilhot, B. Monsarrat, and L. Mourey (IPBS) for fruitful discussions. We also thank C. Houssin and C. de Sousa (University of Paris XI, Orsay, France) for sharing unpublished data. Supplementary Material Download .pdf (3.01 MB) Help with pdf files Download .pdf (3.01 MB) Help with pdf files" @default.
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• W2022719417 title "The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis" @default.
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