Further Insight into S-Adenosylmethionine-dependent Methyltransferases

Abstract

Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 Å. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially theα2-α3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived. Mycolic acids are major and specific components of the cell envelope of Mycobacteria that include Mycobacterium tuberculosis, the causative agent of tuberculosis. Their metabolism is the target of the most efficient antitubercular drug currently used in therapy, and the enzymes that are involved in the production of mycolic acids represent important targets for the development of new drugs effective against multidrug-resistant strains. Among these are the S-adenosylmethionine-dependent methyltransferases (SAM-MTs) that catalyze the introduction of key chemical modifications in defined positions of mycolic acids. Some of these subtle structural variations are known to be crucial for both the virulence of the tubercle bacillus and the permeability of the mycobacterial cell envelope. We report here the structural characterization of the enzyme Hma (MmaA4), a SAM-MT that is unique in catalyzing the introduction of a methyl branch together with an adjacent hydroxyl group essential for the formation of both keto- and methoxymycolates in M. tuberculosis. Despite the high propensity of Hma to proteolytic degradation, the enzyme was produced and crystallized, and its three-dimensional structure in the apoform and in complex with S-adenosylmethionine was solved to about 2 Å. Thestructuresshowtheimportantroleplayedbythemodificationsfound within mycolic acid SAM-MTs, especially theα2-α3 motif and the chemical environment of the active site. Essential information with respect to cofactor and substrate binding, selectivity and specificity, and about the mechanism of catalytic reaction were derived. Mycolic acids, α-branched β-hydroxylated long chain fatty acids, are the hallmark of the Mycobacterium genus that comprises the causative agents of human diseases such as tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. These major cell envelope components play an important role in the structure and function of the mycobacterial cell envelope (1.Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar, 2.Goren M.B. Brennan P.J. Youmans G.P. Tuberculosis. W. B. Saunders Co., Philadelphia, PA1979: 63-193Google Scholar). For instance, mycolic acids attached to the cell wall arabinogalactanareorganizedwithotherlipidstoformanouterpermeabilitybarrier with an extremely low fluidity that confers an exceptional low permeability to mycobacteria and may explain their intrinsic resistance to many antibiotics (3.Brennan P.J. Nikaido H. Annu. Rev. Biochem. 1995; 64: 29-63Crossref PubMed Scopus (1563) Google Scholar). Similarly, trehalose mycolates have been implicated in numerous biological functions related both to the physiology and virulence of M. tuberculosis (1.Daffé M. Draper P. Adv. Microb. Physiol. 1998; 39: 131-203Crossref PubMed Google Scholar). Numerous studies have been and are currently devoted to understanding the structures and biosynthesis of mycolic acids, primarily because they are specific to the Mycobacterium genus, and their metabolism is the only clearly identified target inhibited by the major anti-tubercular drug isoniazid (4.Winder F.G. Collins P.B. J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (174) Google Scholar, 5.Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (230) Google Scholar, 6.Davidson L.A. Takayama K. Antimicrob. Agents Chemother. 1979; 16: 104-105Crossref PubMed Scopus (49) Google Scholar, 7.Quémard A. Lacave C. Lanéelle G. Antimicrob. Agents Chemother. 1991; 35: 1035-1039Crossref PubMed Scopus (106) Google Scholar, 8.Rozwarski D.A. Grant G.A. Barton D.H. Jacobs Jr., W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (613) Google Scholar). With the resurgence of tuberculosis infections caused by multidrug-resistant strains and the need for the development of new anti-tubercular drugs, deciphering the biosynthesis pathway leading to mycolates still represents a major objective. Although much work remains to be done to complete their biosynthetic scheme, it is known that two mycobacterial fatty acid synthases participate in the formation of all types of mycolates. Fatty acid synthase I is necessary to produce C16,18 and C22-26 saturated fatty acids, which may be either directly incorporated into mycolates as the α-branch chain or used as substrates of the acyl carrier protein-dependent elongation system, fatty acid synthase-II, to produce the long meromycolic chain (for a recent review, see Ref. 9.Takayama K. Wang C. Besra G.S. Clin. Microbiol. Rev. 2005; 18: 81-101Crossref PubMed Scopus (506) Google Scholar). Mycolic acids usually occur in mycobacterial species as a mixture of various related molecules with different chemical groups at the so-called “proximal” and “distal” positions of their meromycolic chain of the M. tuberculosis complex tuberculosis, Mycobacterium Mycobacterium Mycobacterium and Mycobacterium types of mycolates are M. Lanéelle C. David H. Microbiol. Scholar, M. M. Microbiol. PubMed Scopus Google Scholar). I are of fatty acids Lanéelle C. B. M. PubMed Scopus Google and two and mycolates of Lanéelle C. B. M. PubMed Scopus Google and a or a methyl group the group at the and a or group a methyl group the at the These structural variations in mycolates may be of crucial biological it been that in the of these chemical and the and the permeability of the cell envelope to and the virulence and of the strains in infections Microbiol. 1998; PubMed Scopus Google Scholar, Jacobs Jr., W.R. PubMed Scopus Google Scholar, J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar, J. 2005; PubMed Scopus Google Scholar). the that the chemical modifications in the mycolic acid chain of the that mycolic acid S-adenosylmethionine used S-adenosylmethionine-dependent acyl carrier M. methyltransferases in M. tuberculosis J. C. K. K. K. K. 1998; PubMed Scopus Google the A. PubMed Scopus Google or J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google been to be necessary and for the of both keto- and acids. of the in the of the of both types of mycolic acid Lanéelle C. B. M. PubMed Scopus Google Scholar, J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar, P. C. A. Lanéelle Lanéelle G. M. J. PubMed Scopus Google Scholar). of Mycobacterium with the in the production of of acids with an adjacent methyl related to the acids of M. tuberculosis Lanéelle A. J.C. M. A. Microbiol. PubMed Scopus Google Scholar). of these acids were in mycobacterial species keto- the that acids be the of both keto- and acids A. Lanéelle H. M. J. Biochem. PubMed Scopus Google Scholar). of the mycolic acids by the that the a new of in which a a P. C. A. Lanéelle Lanéelle G. M. J. PubMed Scopus Google Scholar). the Hma a methyl group the cofactor or a the of leading to the formation of an J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, Lanéelle A. J.C. M. A. Microbiol. PubMed Scopus Google Scholar, A. Lanéelle H. M. J. Biochem. PubMed Scopus Google that methyltransferases meromycolic Microbiol. 1998; PubMed Scopus Google Scholar, Takayama K. J. PubMed Google Scholar). the mechanism of of reaction and as a for drug have the Hma and solved its three-dimensional structure in the and in complex with to and of respectively. of the Mycolic of M. M. tuberculosis was by and into the of the the the the the and a at the leading to a that acid as a the or together with the was by and into the and of the mycobacterial M. was by L.A. G.P. Jacobs Jr., W.R. 1991; PubMed Scopus Google with the or strains were with and were used to the were to as Lanéelle C. B. M. PubMed Scopus Google Scholar). the were by with in mycolic acid methyl were used as M. Lanéelle C. David H. Microbiol. Scholar). Fatty acid methyl were by acid in and and of Hma for used for and was by the into the and of the the the the and a at the leading to a that acid of the Hma was in were at in with was for cell an of and cell were for at were by at for at and the were with and at were by and at for at Hma was by an the was to a with in the was with in the to the were and and the was by of at and were by with a and both with 2 were to and at was by at and was with a For were with of was as in and J. W. PubMed Scopus Google and B. 2: PubMed Scopus Google Scholar). the were and with and the were to of the at by in of and were the by a to the the was in and with were in of and the were by with and were with was an of in of was to the in the and were and of the the and was the at of at in were and the was at were a of was the target with of in were in to and with were with the of the of and was at by the were to in the were by of and of were and low were used for of Hma were for the in the of were in a of at a in the with and in For of the complex with the were in a both the and S-adenosylmethionine for various were at and a at and and a used for structure and were at of the and were the PubMed Scopus Google as in the P. M. PubMed Scopus Google Scholar). were of the of and and with of the and and Scholar). structure of Hma in its was solved with the PubMed Scopus Google and the structure of Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google was as a for the of mycolic acid M. tuberculosis. structure of Hma in complex with its cofactor was solved the of and with the to for the cell and of was with the A. PubMed Scopus Google Scholar). used for the to the of the with different were structures were in A. Scopus Google of the structures were with the PubMed Scopus Google a the and a target molecules were as A. PubMed Scopus Google Scholar). the of were a group for the which in a of the and of the was produced the P. M. PubMed Scopus Google Scholar). and were produced J. Scholar). was PubMed Scopus Google Scholar). was produced P. 1998; Scopus Google with a and structure with P. 1995; PubMed Scopus Google Scholar). were produced mycolic acid of the of Hma and structures are by and active of mycolic acid of the chemical environment of the cofactor and of a as in the structures of and and and of all are for of molecules of Hma are in for M. tuberculosis mycolic acid were to by of with respect to Hma of are in are as a structure for and for of Hma in complex with and of in complex with and are at the structure that participate to the are in of that with the of Hma that be proteolytic are of the motif of mycolic acid and of the motif of were the chain of are in chain of or are in and respectively. of that the group is in motif of and the and of the are as and of the mycolic acid is a the motif the the in and is in and within of the are in directly the were represent in the motif of mycolic acid and the structures of and have been as the are and of was both into and of a and these were used to and respectively. Hma were these strains and to by and by the the and at leading to was for the and which used of the and at to the of two that these two they a within a different and were in in the of and Hma and of the a These that the two of Hma were to these were the and the were and by For the and the is in with the of Hma the the the and of Hma and the of the of to and were and 2 respectively. the of these two and the for Hma may to the formation of the that and a of the were of in and of and 2 the of and respectively. of and 2 the specific of a unique at as the of the and the of to and the and of the Hma of to and and of to and the of a specific proteolytic and by at the of and acids and a to the of Hma to and were both to the specific of of the PubMed Google Scholar). of to the of a proteolytic the with and to which is to of the was in with 2 and which a much of the that a the function of Hma in within For M. was with either the or together with a a that the mycolic acid of both strains was that the of the at the of the Hma its function and that to was with the and of and were respectively. of these two for studies were with Hma in in the of and and were at in the these only by their the and cell complete were to Hma in its and in complex with S-adenosylmethionine to and respectively. of the are in of Hma were to the group and is a of and a of and cell in are for the unique in are for the in a new structure of was solved and the three-dimensional of Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google as a unique was with of the for the and functions of and and a of structure of was used as a for the of to for the cell structure of comprises of acids in the and are and for all and of comprises acids, and the and are and for all and Å. structures have of the in the most of the and in the as defined by J. Google Scholar). defined and to the in both and to a only in the structure of the of a to the in are for the of into the to the of of of cofactor of of in are for the in are for the that include the the that include the the in are for the into the to the that include the the in a new of structure of Hma of the so-called SAM-MT (for a review, see Ref. PubMed Scopus Google with an of and in a in the all are by which is long and an in known structures of mycolic acid SAM-MTs, is extremely in with only two and is to a of the at positions which a and a that is with These molecules are for the chain of and and the chain of in the to both of the are in and about the for the and which is in the of the PubMed Scopus Google comprises that of the in all mycolic acid SAM-MTs, SAM-MTs, Hma variations to the in mycolic acid PubMed Scopus Google these variations to and and to the of a and and to of two long both and and and be that and are only of the cofactor and in the structure of an active of the the of the acid of Hma comprises at positions and of which and are mycolic acid These are at the of the in the of and and of and are and in a is the of Hma participate in the of that the structure of the is and is mycolic acid SAM-MTs, is only in the of with an in all of Hma of a of the the of a specific that and which both that the as a in of the and of of the and structures to a of for the occur in to and these the an of for at the of Hma a different with in the structure of the which is its the cofactor are to the and a that the two and and are of the two the and comprises that be in the of the was clearly in both a major structural these and the Among the that in the two structures of only is to for binding, the a of the of all Mycolic structures of involved in mycolic acid biosynthesis have been Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google in its and in complex with the cofactor and either or the complex of with and and the complex of with three-dimensional of in complex with and were in the These structures and the two of Hma were a complex that have to it to be that groups of structures be the structures of both of of for the structures of the of and and the structures of and the of to and the most structure that of We for specific and that they were to a of of which have been the two structures of of at the which were to be in the structures of or of with either the cofactor substrate or the cofactor to as by the for to and its to the and in are of to or and of to the and which a in the structures of Hma and it a in in all and is the and most in of structural the to the and and to a to be protein-dependent related to the different of a the two structures of Hma to such a of in the of known structures the for the structures of the and of the may be an that and is specific to the which structures of all in the at the of the and and which is to Mycolic acid of of in a both the and for was to of the of the cofactor substrate to Hma in the as the for the cofactor in the structures of the different of Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google and which were by a the of the and to is by of which are directly involved in with the the a structural binding, and and as to the and and and to the of and and to and and These are in the of mycolic acid SAM-MTs, for which is by an in and of in identified Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of and all mycolic acid SAM-MT that both molecules with the leading to of about for the methyl of of the cofactor molecules are in all structures with formation of at to the cofactor to the of the acids that the a the of a in and of a in in in are at of the acid and of the respectively. in the structures the hydroxyl group of of is at of the cofactor methyl group in it is that the the in the of the and substrates because of with and acid their either or Microbiol. 1998; PubMed Scopus Google Scholar, Takayama K. J. PubMed Google Scholar). These substrates are by the fatty acid synthase K. Adv. Google in the of the acyl are to the group of the acyl carrier M. tuberculosis in to and the mycolic acid have the to with and a long acyl chain to the about these specific have been by the structures of the of and with and Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). these structures is a the to the cofactor site. of the is by the long that comprises of and and the that and the be to as the motif of Hma that be with its with as and for and Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). of which two of and at the of the respectively. These two are by a with a the of to the Although these to be all the mycolic acid of known structures the the structure of been solved by G. J. J. PubMed Scopus Google Scholar). the in of the at its the to which the group is attached and is for the of with its as as the of the an that the and for mycolic acid and the of and the is by the which a with the the of the of the is in both structures of Hma to the by and in and with the chain of the the structures of and of in the the of the of their are in to the and and especially to the chain of the of and that the of the is with the structure of in complex with the cofactor it is the of and the of that the in Mycolic of mycolic acid structures in the specific that they be into both to the and to the in the as in the of and in the of and and in the of is that the and the of the and are within a the of of Hma is by with of and and by two with respect to structural of to the of the in of Hma a proteolytic be to a with a substrate for been that the of mycolic acid and the be a structural of by the to the active the reaction at the Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). Hma is a of enzyme for the production of acids J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, Lanéelle A. J.C. M. A. Microbiol. PubMed Scopus Google and are for the introduction of in Jacobs Jr., W.R. PubMed Scopus Google and mycolates Jacobs Jr., W.R. J. PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google respectively. the function of is it been to be for introduction of the in J. PubMed Scopus Google and of the in mycolates A. PubMed Scopus Google Scholar, Lanéelle A. J.C. M. A. Microbiol. PubMed Scopus Google Scholar). the structural in the motif only be related to the of mycolic acid play an important role with respect to their Mycolic acids are important components of M. tuberculosis. Their which are by chain and chemical in the of of against the environment of the For instance, the of the of long in Jacobs Jr., W.R. PubMed Scopus Google Scholar). of keto- and methoxymycolates to of the M. tuberculosis in the of J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar). to the virulence and the structural and the variations in mycolates may be of crucial biological with respect to the permeability of the cell envelope to J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar, A. M. Microbiol. PubMed Scopus Google Scholar, J. Besra G.S. Nikaido H. J. PubMed Scopus Google Scholar, M. C. Lanéelle C. C. B. M. Microbiol. PubMed Scopus Google Scholar). the of the enzymes involved in the chemical modifications of mycolates in new antitubercular drugs effective against M. tuberculosis, strains. which role in mycolic acid K. Wang C. Besra G.S. Clin. Microbiol. Rev. 2005; 18: 81-101Crossref PubMed Scopus (506) Google Scholar, J. PubMed Scopus Google all mycolic acid three-dimensional structures known are of the synthases and known as is unique in that it is the enzyme for the production of the for all mycolates in M. tuberculosis J. C. Lanéelle K. A. M. Microbiol. PubMed Scopus Google Scholar, Lanéelle A. J.C. M. A. Microbiol. PubMed Scopus Google Scholar). produced by Hma be either into by the J. PubMed Scopus Google Scholar, H. M. A. Microbiol. 1998; Google or into by an a which been mechanism by which Hma the introduction of the methyl branch and adjacent hydroxyl is for the mycolic acid it been to a high methyl group to A. PubMed Scopus Google and long chain are the substrates of Hma P. C. A. Lanéelle Lanéelle G. M. J. PubMed Scopus Google Scholar). been that mycolic acid catalyze the a such as a that be by the of two in an biosynthesis pathway C. J. Lanéelle G. Lanéelle PubMed Scopus Google Scholar). With respect to cofactor binding, the structures of Hma that the the to the complex with the substrate a major the of leading to the formation of and subtle structural variations of structures of Hma that of the of mycolic acid SAM-MTs, to the acid to the of the the the structural the that the of mycolic acid need to a for the enzymes to for the of the and the substrate as Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). substrate and the the structural that mycolic acid in their or in complex with are for of their the may in a and an of the with as the structures of the of synthases with and or Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). of the is by the motif that to the specific of mycolic acid motif the mycolic acid and play a role the specific function of of these that the structure of the motif be into that to the known in the of in the of mycolates and in the of and in the of role by the motif for such selectivity and be related to in the of the that the meromycolic chain to C. J. Lanéelle G. Lanéelle PubMed Scopus Google Scholar). it may explain the the of the different mycolic acid Jacobs Jr., W.R. PubMed Scopus Google Scholar, Jacobs Jr., W.R. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). been that the high the of an the methyl group to the formation of a the of an hydroxyl group to the formation of A. PubMed Scopus Google Scholar, Jr., Microbiol. Rev. PubMed Scopus Google Scholar). the of a that a in the of synthase and the of a or in the active of which may the the for instance, a for a been in the active of and that for as the necessary to complete reaction Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). role by in the of the formation of been for the related fatty acid synthase A. J. 2005; PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). the of the is the methyl of as the that of the the methyl group is and in the active site. was in the group of and the which is in is in all may function as a Jacobs Jr., W.R. Sacchettini J.C. J. PubMed Scopus Google Scholar). the active of Hma the of a of the of and and and two is to the of which in with the group of is to the groups of both and These two molecules are and the of as in the is mycolic acid SAM-MTs, in a is is of the and that be involved in is and and structural the propensity of Hma to proteolytic in is is to the report of such an for a mycolic acid is that such a of the enzyme occur in and it be related to a or a it be of been for fatty acid synthase is for the of J. Jr., J. PubMed Scopus Google Scholar). We are to and Lanéelle for and for We for the of work and for in the We the of for