The Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

A closely related family of enzymes fromMycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived fromS-adenosyl-l-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-l-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain. A closely related family of enzymes fromMycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived fromS-adenosyl-l-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-l-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain. Mycobacterium tuberculosis replicates within the hostile environment of the mammalian macrophage whose microbicidal products are generally insufficient to kill this highly resistant bacillus. This intrinsic resistance to antibacterial substances is due, in large part, to the impermeable nature of the mycobacterial cell wall (1Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1524) Google Scholar, 2Liu J. Barry III, C.E. Nikaido H. Ratledge C. Dale J.W. Mycobacteria: Molecular Biology and Virulence. Chapman and Hall, 1998: 1-44Google Scholar, 3Barry, III C.E. Lee R.E. Mdluli K. Sampson A. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; (in press)Google Scholar). The mycobacterial cell wall consists of three covalently attached polymers: the peptidoglycan, the arabinogalactan, and the mycolic acids. The very low fluidity of the hydrophobic domain of this structure significantly reduces the rate at which hydrophobic substances are taken in and thereby potentiates the toxicity of such substances. This low fluidity is directly attributable to the structure of the mycolic acids, which comprise a large proportion of the cell wall mass (4Liu J. Barry III, C.E. Besra G.S. Nikaido H. J. Biol. Chem. 1996; 271: 29545-29551Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Because of the essential nature of this structure to the intracellular life of M. tuberculosis, its biosynthesis and assembly offer critical potential targets for chemotherapeutic intervention. Mycolic acids are α-alkyl-β-hydroxy fatty acids of exceptional length and complexity, which range up to 80 carbons in total chain length and include various functional groups such as cis ortrans cyclopropanes or olefins, α-methyl methyl ethers, or α-methyl ketones. Because of their structural complexity and issues of overlap with enzymes responsible for synthesizing short-chain fatty acids, in vitro systems for studying their biosynthesis have been difficult to develop. We have identified a family of six homologous enzymes by heterologous expression in the saprophytic mycobacterial species Mycobacterium smegmatis that are involved in generating structural diversity among these molecules by the apparent addition of a methyl group fromS-adenosyl-l-methionine (SAM) 1The abbreviations used are: SAMS-adenosyl-l-methioninePCRpolymerase chain reactionHPLChigh performance liquid chromatographyFAB-MSfast atom bombardment mass spectroscopy.1The abbreviations used are: SAMS-adenosyl-l-methioninePCRpolymerase chain reactionHPLChigh performance liquid chromatographyFAB-MSfast atom bombardment mass spectroscopy. (5Yuan Y. Lee R.E. Besra G.S. Belisle J.T. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6630-6634Crossref PubMed Scopus (169) Google Scholar, 6George K.M. Yuan Y. Sherman D.R. Barry III, C.E. J. Biol. Chem. 1995; 270: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Yuan Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (127) Google Scholar, 8Yuan Y. Crane D.C. Musser J.M. Sreevatsan S. Barry III, C.E. J. Biol. Chem. 1997; 272: 10041-10049Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The mechanism of methylation by these enzymes has been proposed to involve the formation of an intermediate carbocation, which can then react to form a variety of different chemical structures depending upon the active site configuration of the particular enzyme resulting in the observed structural diversity of mycolic acids. S-adenosyl-l-methionine polymerase chain reaction high performance liquid chromatography fast atom bombardment mass spectroscopy. S-adenosyl-l-methionine polymerase chain reaction high performance liquid chromatography fast atom bombardment mass spectroscopy. Although heterologous expression studies have proven useful for identifying the genes encoding the enzymes involved in mycolic acid modification, they have not resolved critical issues regarding the substrate for methyl(ene) group transfer, including lipid chain length during SAM addition and the head group or acyl carrier moiety during methyl addition. Previous reports of radiomethyl group addition from SAM to very long-chain (∼48–56 carbons) mycolate precursors by cell-free supernatants of M. tuberculosis H37Ra suggested that development of a cell-free system based upon combining recombinant enzyme produced in Escherichia coli with soluble acceptor fractions from various mycobacterial species would prove successful (9Qureshi N. Sathyamoorthy N. Takayama K. J. Bacteriol. 1984; 157: 46-52Crossref PubMed Google Scholar). In addition, we have recently demonstrated that the long meromycolic acid chain is synthesized on an acyl carrier protein designated AcpM by a novel type II fatty acid synthase system (10Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (372) Google Scholar). To understand the biosynthetic details of meromycolate modification and the relationship of this system to the AcpM-utilizing type II fatty acid synthase system, we developed cell-free systems for studying the transfer of a methyl group by the meromycolate methyl transferase enzyme family. M. tuberculosis strain H37Rv (ATCC 27294), M. smegmatis mc2155 (provided by William R. Jacobs, Albert Einstein College of Medicine, New York) and the various recombinants were grown at 37 °C in Middlebrook 7H9 medium with albumin/dextrose/catalase supplement (ADC) containing, where appropriated, kanamycin (25 μg/ml) (Sigma) or hygromycin 50 μg/ml) (Calbiochem). E. coli strain DH5α and JM109 (Life Technologies, Inc.) were used for routine DNA manipulations and grown in LB kanamycin or hygromycin μg/ml) or (Sigma) The cells of E. coli strain (ATCC with DNA S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google were by at °C in LB with kanamycin in the was and by and in were in of and its were in and at In addition to the with and Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (127) Google were produced from the AcpM, and were used to is shown in These site at the of the and site the The was in was then from this and the of to the expression coli strain with was grown at 37 °C in LB to an of then with for at The mass of the domain protein produced by is The of genes for the and were from the by with DNA polymerase the of The products were with the for for for and with also with in either DH5α or with were and were then used to strain S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google for as expression in M. and were as K.M. Yuan Y. Sherman D.R. Barry III, C.E. J. Biol. Chem. 1995; 270: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (155) Google Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (127) Google Scholar). and DNA were from New or DNA were and with or cell were from of M. smegmatis cells grown to an of The cell were with of and at at °C for The cells were in of and to a with of in The cells were by for with an on The cell was at at °C for and then the was heat-inactivated at °C for In the was used on the In substrate was from substrate was with that of Overproduction of recombinant in was by cells from an grown in of LB at °C to of The was then with at °C for by 37 °C for with E. coli cells were and as for mycobacterial of substrate and enzyme coli were in a and with of at 37 °C for The were in at °C with was as K.M. Yuan Y. Sherman D.R. Barry III, C.E. J. Biol. Chem. 1995; 270: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The methyl esters were by an to by and was by a N. Takayama K. J. Biol. Chem. Full Text PDF PubMed Google K. N. of New Scholar). were methyl esters from fatty acids the cell-free the was to at The shown in was to to In the were with (Sigma) or J. Scopus Google or at 37 °C for the addition of and was to to the mycobacterial and the were and at °C The substrate was by a The of the (or to and was by the reaction with at 37 °C by the addition of an of or by for by and to produce an and an The of methylated of was by A (10Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (372) Google was and used to AcpM is the carrier of the substrate for The from the cell-free reaction was and then with The soluble were on a that was developed with acid of with different were from the and with The of was by the of the of [methyl-3H]SAM with of The for the reactions were used to a for of the A. and H. and New Scholar). reaction and the of on the of in and were by the in with different all of these the to all long-chain products were and to total of was with as III, C.E. Science. PubMed Scopus Google Scholar). M. H37Rv was grown at 37 °C to an of at in 7H9 with 80 and The were by at for The resulting cell was in an of and at °C The was then to and an of in was This was on a for at and then to was The resulting was The was with an of then then and to a and at The resulting was in of and of and the full-length mycolic acids were by the addition of of M. J. PubMed Scopus Google Scholar). The was to °C for and then at for The was and the of the mycolic acids was The mycolic acids were then to by with a of and mass of the of mycolic acids were in with that Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (127) Google Scholar). The meroaldehyde of the various was as J. Scopus Google Scholar). mycolic acid was in a reaction a of of and to °C for The resulting meroaldehyde was on a an to A. C. PubMed Scopus Google Scholar). The meroaldehyde was oxidized by the addition of in a of and at for The resulting was with acid and three with The was at 50 The resulting the acid was in and as (5Yuan Y. Lee R.E. Besra G.S. Belisle J.T. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6630-6634Crossref PubMed Scopus (169) Google Scholar). The methyl mycolate was by The was then at in a of in at for The was then with acid and with three The was and to III, C.E. Science. PubMed Scopus Google We had six different enzymes involved in modification reactions of mycolic acids by heterologous expression in M. smegmatis and M. tuberculosis. the (5Yuan Y. Lee R.E. Besra G.S. Belisle J.T. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6630-6634Crossref PubMed Scopus (169) Google the K.M. Yuan Y. Sherman D.R. Barry III, C.E. J. Biol. Chem. 1995; 270: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (155) Google a to with the of an methyl Y. Crane D.C. Musser J.M. Sreevatsan S. Barry III, C.E. J. Biol. Chem. 1997; 272: 10041-10049Abstract Full Text Full Text PDF PubMed Scopus (82) Google and and produce acids Y. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12828-12833Crossref PubMed Scopus (127) Google Scholar). To the of these enzymes in we of E. coli expression which either a at the or a domain and at the expression of of these genes was with the of a protein of the as The of the of and was by to to a to of this family of enzymes not of an in M. smegmatis and of the mycolic acids produced by this strain the presence of the mycolic acid (5Yuan Y. Lee R.E. Besra G.S. Belisle J.T. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6630-6634Crossref PubMed Scopus (169) Google that the presence of the not enzyme activity not We also of these enzymes in M. an expression system based on the and K.M. Yuan Y. Sherman D.R. Barry III, C.E. J. Biol. Chem. 1995; 270: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In M. of enzyme was and the of mycolic acids from these was as To various in vitro we the transfer of from [methyl-3H]SAM to long-chain fatty acids in of M. tuberculosis, which had been by Takayama and (9Qureshi N. Sathyamoorthy N. Takayama K. J. Bacteriol. 1984; 157: 46-52Crossref PubMed Google Scholar). we then type M. smegmatis and and methyl no long-chain we observed by an at this to the of methylation of acid C. Ratledge C. J. The Biology of the Scholar). This activity be by the M. smegmatis to °C for E. coli of the enzymes no of but with or from M. smegmatis the recombinant the of a from SAM very long-chain lipid with and The and of the were critical to substrate of the M. smegmatis at °C for to addition of the recombinant activity not heat-inactivated acceptor °C for from M. smegmatis and recombinant produced in E. we were to cell-free activity from and and produced produced mycolate precursors that by The of this are with the of a the meromycolate and the presence of the large domain not the of this protein to its not in of with the of an on heterologous expression of this enzyme in M. smegmatis Y. Crane D.C. Musser J.M. Sreevatsan S. Barry III, C.E. J. Biol. Chem. 1997; 272: 10041-10049Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). tuberculosis the of long-chain precursors with the recombinant with the not also to in vitro with the that this enzyme the produced by and with M. smegmatis acceptor produced of long-chain with the and the other significantly the methyl of these by that the of lipid was suggesting of such precursors are in We also cell-free methyl(en)ation of mycolate precursors in of recombinant M. smegmatis various enzymes not In this activity was that observed by the enzyme and substrate This system was useful for chain length was difficult of the potential for of molecules by the fatty acid synthase II system, and of the substrate was by the presence of enzyme and substrate in the In vitro methyl(ene) transfer be inhibited by low the of SAM This level of was to that with other PubMed Scopus Google and was also observed with SAM M. PubMed Scopus Google Scholar). of whole cells of either M. tuberculosis or various smegmatis with up to of these not the mycolic acids produced they inhibit The reaction was found to be the and the apparent for SAM was as The reaction was found to be very with a in activity and This in activity was found to be with a in of the reaction with and the of the at basic and the of the was with the The this and fatty acids. The soluble as as and products at either the was and methyl The in soluble at basic is with of a of the lipid of the substrate to a carrier a The enzyme produced in M. smegmatis to be soluble by of soluble and fractions that with either or not The substrate for methyl transfer also to be activity be observed with of the with to which with of the protein family a for the were at in This the were in M. suggesting fatty acid synthase from E. these enzymes are with a with systems E. J. PubMed Scopus Google of mycolate smegmatis were with to the which with other of this protein family. of were by as either not with cells with the or with the cell wall of the The results of with M. smegmatis are These reaction with the of the with M. smegmatis no as with an in a were with to the which with other of this protein family. of were by as either not with cells with the or with the cell wall of the The results of with M. smegmatis are These reaction with the of the with M. smegmatis no as with an The cell-free methyl(en)ation reaction to the of the length of the lipid during this of the a relationship chain length and as has been by N. Takayama K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. N. of New Scholar). from the fatty acid carbons in was to that the lipid by in the cell-free system with a of a of The of a the lipid would have the effect of the the apparent chain by depending upon the of the group (9Qureshi N. Sathyamoorthy N. Takayama K. J. Bacteriol. 1984; 157: 46-52Crossref PubMed Google Scholar). meromycolate tuberculosis would be to have the of such as as methyl or the difficult to mycolic with an group and an from this at 50 To the of the of the and long-chain mycolate we synthesized meromycolic acid from full-length mycolic acids from the bacillus. of full-length mycolate has been and was used in the structural of these molecules III C.E. Lee R.E. Mdluli K. Sampson A. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; (in press)Google Scholar, A. J. Biol. Chem. Full Text PDF Google Scholar). a short-chain fatty acid identified as acid and the long-chain The from the the by and a of by of the of this was an of basic also as J. Scopus Google Scholar). The of this was to a methyl and and the by the as for the transferase reaction this was shown to at the full-length mycolic acid and with the of the observed in the in vitro system and The of and to an extremely of that are potential for methyl In addition, the of the chain length of these during the of has not been This of lipid was different enzymes on the substrate A the a the of the was with acceptor fractions from heat-inactivated M. smegmatis and The used to these is in the long-chain the to the complexity of the lipid of the in these is of a carbons in length and to full-length meromycolate a the a with chain the that the occur to the of the which occur the chain is at a carbons in these results the that meromycolate modification occurs with meromycolate This formation is not as apparent with of chain in fatty acids of length are in systems and fatty esters of carbons are also highly the for these methyl transfer reactions to be soluble To understand the nature of the carrier that these to we a of to the of this the carrier was sensitive to at °C for but was resistant to °C for We also found that of the substrate with by the addition of not destroyed the to such In addition, with by with by to in all of the the in the The substrate be by and activity The substrate also be by fast protein liquid The was with a and the results suggested that the carrier was in We recently identified an acyl carrier AcpM, which with attached acid in the presence of and to in to full-length meromycolate in the of (10Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (372) Google Scholar). To AcpM was the carrier during methyl(ene) transfer in these in vitro reactions, we of M. tuberculosis with the of AcpM and then with had no effect on the of such to long-chain but with inhibited such reactions in were by in the acid system in which we have the was found to comigrate with with of of a family of whose heterologous expression in the modification of full-length mycolic acids the of the of these enzymes but regarding the details of the biosynthetic to these cell wall III C.E. Lee R.E. Mdluli K. Sampson A. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; (in press)Google Scholar). these details is essential these reactions are to be as potential chemotherapeutic In addition, of active enzymes was essential for structural studies of these enzymes to the that a of active site and produce chemical diversity among mycolic acids. a all six of these enzymes have been in E. coli as and an in vitro has been developed to that these enzymes activity as In vitro methyl transfer from to an acceptor from the mycobacterial has to the lipid length of the meromycolate chain during modification as as the carrier for chain a which this with the from an of the mechanism of of (10Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (372) Google Scholar). In this for the type II fatty acid synthase system, which mycolic acids, are produced by the type fatty acid These are and These are then A to the acyl carrier protein The various molecules are by the type II system which include and A. E. A. D. Science. PubMed Scopus Google Scholar). The carbons of the acid not to be synthesized they are modification synthesis and occurs the acids are attached to the These studies not the at which occurs to the chain and occurs in a parallel or to a substrate that is then by the type II In these heat-inactivated the of the type II system are not of the length of the the length of the substrate This has the that the various and enzymes are the chain from the that they from the but have a very substrate The that the in vitro reaction can be inhibited by for AcpM and that the acceptor has the of an that the acid attached to this during modification and for this from an of potential in the mycobacterial R. J. K. S. Barry III, C.E. 1998; PubMed Scopus Google in which no of a that fatty acids. the potential homologous to enzymes on but not homologous to enzymes on These results also the of Takayama and identified of and potential meromycolate from carbons in length (9Qureshi N. Sathyamoorthy N. Takayama K. J. Bacteriol. 1984; 157: 46-52Crossref PubMed Google Scholar, N. Takayama K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. N. of New Scholar, K. Rev. Google Scholar, K. N. PubMed Scopus Google Scholar). The nature of the carrier during the reaction has not been identified R.E. J.W. Takayama K. P.J. Besra G.S. 1997; PubMed Scopus Google we have that this is to be a III C.E. Lee R.E. Mdluli K. Sampson A. Schroeder B.J. Slayden R.A. Yuan Y. Prog. Lipid Res. 1998; (in press)Google Scholar). The to the acid of these also the details of the to be reports have of a system of synthesizing from not from C. A. PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar, A. C. PubMed Scopus Google Scholar, Besra G.S. Ratledge C. PubMed Scopus Google Scholar). These reports have suggested that all of the enzymes for mycolic acid are with a system through a of is difficult to the of this system of but not or would be for an fatty acid synthase system, in the meromycolic acid modification enzymes not this fraction, E. Barry III and R. A. in such fractions of or that very and (4Liu J. Barry III, C.E. Besra G.S. Nikaido H. J. Biol. Chem. 1996; 271: 29545-29551Abstract Full Text Full Text PDF PubMed Scopus (230) Google has not been to inhibit such activity by such as The results the that acid synthesis occurs in the cell the fatty acyl chain is covalently attached to AcpM a This has also to in that potential can be directly the reaction be observed by of enzymes such and this for of potential the of novel for in the of tuberculosis and related mycobacterial In addition, structural be structure these recombinant enzyme We Crane for and and for the We and Lee for AcpM and of the as as