A New Family of Type III Polyketide Synthases in Mycobacterium tuberculosis

Abstract

The Mycobacterium tuberculosis genome has revealed a remarkable array of polyketide synthases (PKSs); however, no polyketide product has been isolated thus far. Most of the PKS genes have been implicated in the biosynthesis of complex lipids. We report here the characterization of two novel type III PKSs from M. tuberculosis that are involved in the biosynthesis of long-chain α-pyrones. Measurement of steady-state kinetic parameters demonstrated that the catalytic efficiency of PKS18 protein was severalfold higher for long-chain acyl-coenzyme A substrates as compared with the small-chain precursors. The specificity of PKS18 and PKS11 proteins toward long-chain aliphatic acyl-coenzyme A (C12 to C20) substrates is unprecedented in the chalcone synthase (CHS) family of condensing enzymes. Based on comparative modeling studies, we propose that these proteins might have evolved by fusing the catalytic machinery of CHS and β-ketoacyl synthases, the two evolutionarily related members with conserved thiolase fold. The mechanistic and structural importance of several active site residues, as predicted by our structural model, was investigated by performing site-directed mutagenesis. The functional identification of diverse catalytic activity in mycobacterial type III PKSs provide a fascinating example of metabolite divergence in CHS-like proteins. The Mycobacterium tuberculosis genome has revealed a remarkable array of polyketide synthases (PKSs); however, no polyketide product has been isolated thus far. Most of the PKS genes have been implicated in the biosynthesis of complex lipids. We report here the characterization of two novel type III PKSs from M. tuberculosis that are involved in the biosynthesis of long-chain α-pyrones. Measurement of steady-state kinetic parameters demonstrated that the catalytic efficiency of PKS18 protein was severalfold higher for long-chain acyl-coenzyme A substrates as compared with the small-chain precursors. The specificity of PKS18 and PKS11 proteins toward long-chain aliphatic acyl-coenzyme A (C12 to C20) substrates is unprecedented in the chalcone synthase (CHS) family of condensing enzymes. Based on comparative modeling studies, we propose that these proteins might have evolved by fusing the catalytic machinery of CHS and β-ketoacyl synthases, the two evolutionarily related members with conserved thiolase fold. The mechanistic and structural importance of several active site residues, as predicted by our structural model, was investigated by performing site-directed mutagenesis. The functional identification of diverse catalytic activity in mycobacterial type III PKSs provide a fascinating example of metabolite divergence in CHS-like proteins. Mycobacteria are classified in the phylogeny of the Actinomycetes, along with the Streptomyces bacteria. Interestingly, these two actinomycete genera have received immense attention due to their contrasting effects on human society. Whereas Streptomyces have provided a rich source of antibiotics and other therapeutic products for human diseases, Mycobacterium tuberculosis and Mycobacterium leprae have been two of humankind's greatest scourges. Although the original phylogenic classifications of Mycobacterium and Streptomyces were based primarily on morphology, the genome sequences of these organisms have further established their common lineage (1Cole S.T Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V Eiglmeier K. Gas S. Barry III C.E Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Barrell B.G Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar, 2Bentley S.D. Chater K.F. Cerdeno-Tarraga A.M. Challis G.L. Thomson N.R. James K.D. Harris D.E. Quail M.A. Kieser H. Harper D. Bateman A. Brown S. Chandra G. Chen C.W. Collins M. Cronin A. Fraser A. Goble A. Hidalgo J. Hornsby T. Howarth S. Huang C.H. Kieser T. Larke L. Murphy L. Oliver K. O'Neil S. Rabbinowitsch E. Rajandream M.A. Rutherford K. Rutter S. Seeger K. Saunders D. Sharp S. Squares R. Squares S. Taylor K. Warren T. Wietzorrek A. Woodward J. Barrell B.G. Parkhill J. Hopwood D.A. Nature. 2002; 417: 141-147Crossref PubMed Scopus (2626) Google Scholar). These genome sequences have revealed several families of genes that are common between them, which include an unusually large number of gene clusters that are homologous to polyketide synthases (PKSs). 1The abbreviations used are: PKS, polyketide synthase; CoA, co-enzyme A; CHS, chalcone synthase; 2PS, 2-pyrone synthase; THN, 1,3,6,8-tetrahydroxynaphthalene; THNS, 1,3,6,8-tetrahydroxynaphthalene synthase; ESI, electrospray ionization; MS, mass spectrometry; HPLC, high pressure liquid chromatography. Although a polyketide product has not yet been isolated from M. tuberculosis, recent studies have implicated some of its PKS genes in the biosynthesis of complex lipids (3Kolattukudy P.E. Fernandes N.D. Azad A.K. Fitzmaurice A.M. Sirakova T.D. Mol. Microbiol. 1997; 24: 263-270Crossref PubMed Scopus (163) Google Scholar). In this report, we have characterized two novel polyketide synthases from M. tuberculosis, which are involved in the biosynthesis of unusual long-chain α-pyrones. Polyketides are a diverse class of secondary metabolites that possess a broad range of biological activities (4O'Hagan D. Nat. Prod. Rep. 1993; 10: 593-624Crossref PubMed Scopus (43) Google Scholar). Despite structural diversity of these natural products, PKSs synthesize polyketides by a common chemical strategy. Initial priming by a starter molecule is followed by repetitive decarboxylative condensation of coenzyme A (CoA) analogues of simple carboxylic acids. PKS elongates the polyketide chain either by repetitively using a single active site to perform multiple condensation reactions or by using a modular assembly line mechanism (5Hopwood D.A. Chem. Rev. 1997; 97: 2465-2498Crossref PubMed Scopus (629) Google Scholar, 6Khosla C. Gokhale R.S. Jacobsen J.R. Cane D.E. Annu. Rev. Biochem. 1999; 68: 219-253Crossref PubMed Scopus (301) Google Scholar, 7Katz L. Donadio S. Annu. Rev. Microbiol. 1993; 47: 875-912Crossref PubMed Scopus (317) Google Scholar). Based on protein architecture, PKSs have been classified in three distinct families. Type I PKSs are giant assemblies of multifunctional polypeptides and include both iterative and modular mechanism of biosynthesis. Type II PKSs are a large multienzyme complex of discrete proteins and resemble type II fatty acid synthases found in bacteria and plants. The type III PKS has been recently discovered in bacteria and belongs to the plant chalcone synthases (CHSs) superfamily of condensing enzymes (8Moore B.S. Hopke J.N. ChemBioChem. PubMed Scopus Google Scholar). These are proteins and are and distinct from type I and type II PKSs R.S. D. Scholar). Type III PKSs substrates the of a are in higher and biosynthesis of for J. and of Scholar). to the CHS as 2-pyrone synthase and from the chalcone by either starter or the number of condensation reactions Nat. Prod. Rep. PubMed Scopus Google Scholar). of the plant enzymes the diversity of the CHS The of CHS from Nat. 1999; PubMed Scopus Google and from J. J. Chem. PubMed Scopus Google have to a to the mechanistic between enzymes the thiolase the thiolase enzymes three families Nat. Prod. Rep. PubMed Scopus Google Scholar, Nat. Prod. Rep. 2002; PubMed Scopus Google Scholar, M. K. J. Mol. PubMed Scopus Google Scholar). protein synthase I and II proteins and III and type III PKS The of several These enzymes have a conserved that of the active site The of the catalytic with to the is conserved in this family of enzymes. The primarily in the structural that the other of the The and the number of the catalytic acid might for the in the catalytic The provide a for for other CHS the several CHS have been in bacteria. The type III PKS involved in the biosynthesis of 1,3,6,8-tetrahydroxynaphthalene has been characterized from S. N. M. S. Nature. 1999; PubMed Scopus Google S. J. J. G. Mol. Microbiol. 2002; PubMed Scopus Google S. N. S. Biochem. J. 2002; PubMed Google and S. B.S. C. Hopke J.N. M. G. T. J. L. J. Nat. Prod. 2002; PubMed Scopus Google Scholar). of these synthases as starter and The chain decarboxylative of to which is The from S. as has been to possess broad specificity to a of products N. S. J. Chem. 2002; PubMed Scopus Google Scholar). In several that of natural products, a CHS-like protein the biosynthesis of by using several of as J. J. J. S. J. Chem. PubMed Scopus Google Scholar, H. S. A. PubMed Scopus Google Scholar, H. Chem. Scopus Google Scholar). The to the broad is by a CHS in of J. 1999; PubMed Google Scholar). The protein and two of to which is to In this we have characterized two type III PKS PKS18 and from M. tuberculosis that are involved in the biosynthesis of long-chain α-pyrones. that the catalytic efficiency of PKS18 protein to long-chain substrates is severalfold higher that for small-chain remarkable specificity of mycobacterial proteins is unprecedented in the family of proteins. modeling studies that these proteins might have evolved by the active of CHS and the two related members of the thiolase enzymes. The catalytic and structural importance of several active site residues, as predicted by our structural model, was investigated by performing site-directed mutagenesis. These studies thus a class of in this protein of M. tuberculosis was from of (1Cole S.T Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V Eiglmeier K. Gas S. Barry III C.E Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Barrell B.G Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar). was from and starter substrates were from starter substrates were by was using an and R. S. and III PKS and were by from the of M. tuberculosis using and The of was by These genes were the and as proteins in the of was by Whereas with to of PKS18 in PKS11 protein was in the by for the as and not in the were using and further on a and were using the site-directed was in with the were by and by of the proteins were and to the type PKS18 and as and not in and involved starter molecule and of were with of protein for and with were with of and products were on in acid products were on A of in to in was used for of were using a of and products were characterized using electrospray of of of of and starter molecule were for by the of and for were with of the with The was and on as products were using a of protein was with or for were by the of and on to the proteins was using a of of protein and was for were with and on A of was used to and The of these was using sequences of CHS-like proteins were from D.A. J. 2002; PubMed Scopus Google and of enzymes of thiolase family were from the J. G. H. P.E. PubMed Scopus Google Scholar). were using J. Mol. PubMed Scopus Google and PubMed Scopus Google from the was used for multiple and The active site in were by using the The was used for were using P.E. 1998; PubMed Scopus Google Scholar). were using the of the and the II was used for and of structural of Type III PKS genome of have in identification of several genes to type III A number of CHS are in S. and M. tuberculosis (8Moore B.S. Hopke J.N. ChemBioChem. PubMed Scopus Google Scholar). Although of the type III PKS genome has been these that type III PKS is in bacteria as of mycobacterial genome sequences revealed three type III PKS genes in M. of these and are chalcone synthases, is as a conserved protein in the M. tuberculosis genome C. 2002; PubMed Scopus Google Scholar). The and genes are in an unusual with other PKS genes that of The on the other is not by three genes are conserved in M. their functional in of protein sequences of and type III PKSs have to the of these proteins based on The based on protein sequences two to and further the plant family was not (8Moore B.S. Hopke J.N. ChemBioChem. PubMed Scopus Google Scholar, J. J. G. Mol. Microbiol. 2002; PubMed Scopus Google Scholar). protein diversity between plant and type III PKSs with functional between the two is that the active site in the catalytic active site of from modular PKSs G. Gokhale R.S. D. J. Mol. PubMed Scopus Google and of G.L. J. Chem. PubMed Scopus Google have with their a of and some of the plant type III by using active site that have been in the of The product by the PKS is to its and their products are in the that the plant polyketide synthases The CHS proteins that have been have of active site Interestingly, the plant PKSs with products in the which was not the was with the protein The three distinct of type III PKSs that have been characterized in three distinct we that active site are conserved their family of of active site a of of the sequences are from the are protein sequences in the synthase and of have been predicted with that the involved in which was recently by and Nat. Prod. Rep. PubMed Scopus Google Scholar). protein was as S. genome S. H. J. A. C. M. H. H. T. H. T. M. S. A. PubMed Scopus Google Scholar). from possess an of active site residues, that are involved in the biosynthesis of the The other type III proteins of are in a in the Although several catalytic and structural are these CHS-like enzymes from have several acid not of mycobacterial type III In to the of these mycobacterial type III we have used two distinct In the the of has been by using and R. S. The for the type and Mycobacteria the of PKS The of this In the we have the in the of three mycobacterial CHS The of and PKS18 were by in the catalytic of CHS by in these proteins. the PKS18 was by of that the of The acid were by the using the J. Mol. 1997; PubMed Scopus Google Scholar). comparative modeling is based on the that the as as the active site of protein In CHS was by three J. J. Chem. PubMed Scopus Google Scholar). The in with the in of the starter number of and with synthases have to the structural for Nat. Prod. Rep. PubMed Scopus Google Scholar). The of CHS and synthase has these to active site Although this example the of that the modeling are the for the active site of for mycobacterial type III PKSs with that of the of were for and for The of three and in the due to of these residues, as in its J. J. Chem. PubMed Scopus Google Scholar). to the of active site In the of THNS, these three to and The of and are in of of the for In and of CHS to of is to and Although is a in the of the compared with CHS, of these proteins is not from these In the PKS18 remarkable in the active site The from three active site residues, and the active site We predicted that PKS18 protein might resemble and biosynthesis of In to our for and plant products were in the Most of these in with the protein and that starter not a for these proteins. of Type III mycobacterial type III PKSs with both plant and CHS-like These genes were from the and were in E. using the Although for three and was of the protein was in The PKS11 protein was from and was used to The of PKS18 protein was to by with a PKS11 protein in the by for The from PKS11 protein the and no other were PKS11 was to PKS18 which a of between these two proteins. Despite our protein not in PKS18 and PKS11 proteins were by using acid and were to using chromatography. The of proteins were by protein and mass Although our modeling studies predicted that mycobacterial proteins not to we investigated the of these proteins with several due to their with plant were with PKS18 and PKS11 proteins along with of the by for PKS18 reactions with an from that of the plant with that PKS18 protein synthesize this product from as a starter we PKS18 protein with and the used in this was no in the product PKS11 protein not activity these we to the PKS18 for PKS18 III PKSs as CHS and have been to have starter specificity N. S. J. Chem. 2002; PubMed Scopus Google Scholar, S. A. 2002; PubMed Scopus Google Scholar). in the of has the catalytic both in of of products and in the of PKS18 protein not activity with we used several and long-chain fatty acid as starter of a using several starter and as with PKS18 that several on the for the long-chain is a in the of the products with of the fatty that PKS18 protein is to and long-chain aliphatic as Whereas of PKS18 protein with with long-chain (C12 to C20) two the used in this no activity was for and These studies that PKS18 protein long-chain substrates by using as an molecule to synthesize polyketide of products from enzymes were on and characterized by a of product on we to this The a single a of PKS18 with along with as starter product that with the product was to and a was Based on our of polyketide this mass is for a from molecule of and two of In to products from a was the two products as in mass for and for which from and products, of reactions with from products of type III PKSs are to in three distinct polyketide products are from and of the J. and of Scholar, Nat. Prod. Rep. PubMed Scopus Google Scholar). The structural of PKS18 products was by using mass the for the for which a of to and to has been for the by from S. N. S. J. Chem. 2002; PubMed Scopus Google Scholar). The for a which the of an by PKS18 protein with other were characterized by and to and α-pyrones. The starter specificity of PKS18 was investigated by performing kinetic using PKS18 protein in to the of which is in that the for the biosynthesis of of long-chain substrates are severalfold higher that for small-chain of PKS18 proteins for long-chain as in the is for to substrates were not investigated in this unusual specificity toward long-chain aliphatic fatty is in the CHS family of condensing enzymes. studies have to the starter by CHS and N. S. J. Chem. 2002; PubMed Scopus Google Scholar, S. A. 2002; PubMed Scopus Google kinetic parameters for PKS18 protein with starter in a Based on this unusual of CHS-like proteins to long-chain reactions were with PKS11 protein using and long-chain the of reactions of PKS11 and PKS18 proteins. products were the proteins were for with these and PKS11 these starter and products, as by not of for of the structural of and PKS18 proteins that the CHS active site with aliphatic the of an in these type III PKS proteins from M. is to that some members to the thiolase family possess the to aliphatic as substrates M. K. J. Mol. PubMed Scopus Google Scholar, J.N. G. J. Chem. PubMed Scopus Google Scholar, A. M. S. PubMed Scopus Google Scholar). The of the of III from M. tuberculosis to as has revealed the mechanism by which the decarboxylative condensation of with from the thiolase III and CHS-like proteins the catalytic The structural of on as in a of a of The of the two and the are to the The catalytic with a of is to from that the two active site for CHS and for are with to the Whereas the III the starter to the and toward the CHS toward the CHS, long-chain aliphatic and has been to for to aliphatic substrates J.N. G. J. Chem. PubMed Scopus Google Scholar). The structural of the specificity for priming was investigated recently J.N. G. J. Chem. PubMed Scopus Google Scholar, H. S. J. C. 2002; 10: PubMed Scopus Google Scholar). These studies that the and are primarily by the of three from the to the In of the of the and are either or in a A recent has the structural and mechanistic of members of the thiolase family of enzymes Nat. Prod. Rep. PubMed Scopus Google Scholar). mycobacterial type III PKSs products that have of both CHS and III we to the that these enzymes have evolved by the two catalytic of CHS and PKS18 a of with CHS the protein a comparative of PKS18 based on was which demonstrated a CHS In the structural family of condensing several proteins as long-chain The to long-chain aliphatic in the active site of PKS18 was by using as of PKS18 to is to a of The not with PKS18 in are of structural PKS18 was using the J. Mol. PubMed Scopus Google Scholar). The the plant CHS to the the of was to a of the by the was found that a for due to of the of the from to and thus provided an of PKS18 on the structural of plant CHS was the fold. We to as a for this protein is to long-chain aliphatic the D. of of and of an was in along the that in the active site of the fold. the with are not in the acid was in the active site of PKS18 using the from the of I complex A. M. S. PubMed Scopus Google Scholar). Although I proteins have no to the CHS family of condensing has revealed a conserved thiolase A. M. S. 1999; PubMed Scopus Google Scholar). of the on that the in the of These were used as in the active site of fold. The of that were in with the acid were with of PKS18 based on The of the acid was to the of active site and the complex was using the J. M. PubMed Scopus Google Scholar). The structural of PKS18 with acid is in The in with the a and no were These a structural for the unusual specificity of PKS18 the of PKS18 by has provided a to the functional of catalytic and structural of mycobacterial modeling studies in with the of the specificity for priming that acid from PKS18 a CHS a the which is from the and the active site from that the acid the of the CHS further the The in PKS18 was to of and to the of the was the II specificity was to substrates D. G. K. K. Scopus Google Scholar). of the of PKS18 protein in of these three proteins in Despite our these proteins not in the Although we not the functional of this that a predicted to involved in the of active site of was investigated by performing studies of the in CHS have implicated its in starter and of the chain of the polyketide J. J. Chem. PubMed Scopus Google Scholar). of the to a in protein the of and were N. S. Biochem. J. 2002; PubMed Google Scholar). In was to a characterization with and the starter substrates in a in the of the the mass of the of the of The type PKS18 protein not synthesize this in the product for the that the might in the of PKS18 with our CHS condensing enzymes three conserved acid that are the of the for with Nat. 1999; PubMed Scopus Google Scholar). type III PKSs an conserved acid in in to the predicted Nat. Prod. Rep. PubMed Scopus Google Scholar). have a conserved this as in The is in several III the chain the of protein J. Chem. PubMed Scopus Google Scholar). has been that some type III PKSs have specificity for substrates Nat. Prod. Rep. PubMed Scopus Google Scholar). In an to the of this conserved of PKS18 was to The was to synthesize polyketide products in our PKS18 of the protein no in the secondary and as compared with type PKS18 The of the protein was by with and the of the type PKS18 protein and protein with and that the type PKS18 protein with both and In the the to the starter not to the mechanistic that this protein was in The identification of diverse catalytic activity in mycobacterial type III PKSs an example of divergence in CHS-like proteins. Interestingly, the plant CHS protein has not yet been characterized in bacteria. In this we the characterization of two novel type III polyketide synthases from M. PKS11 and PKS18 enzymes have remarkable specificity to long-chain analogues (C12 to C20) as starter to α-pyrones. The by PKS18 protein is in The mechanism of PKS18 protein to synthesize was to the was a of followed by or this was by The products of PKS18 were with of the that the were not for Although and the of an products with aliphatic of these products in and the of The not or carboxylic acid that the of product by A mechanism of has been for protein N. S. J. Chem. 2002; PubMed Scopus Google Scholar). synthases have been characterized in S. G. J. D. M. J. Nature. 1998; Scopus Google proteins have not been from the plant and type III PKSs are to synthesize as products of a starter N. S. J. Chem. 2002; PubMed Scopus Google Scholar, S. A. 2002; PubMed Scopus Google Scholar). this in Whereas the broad starter molecule specificity of PKS18 and PKS11 enzymes the of in biological the for the type of starter of aliphatic or or or the with the in of the biological The of mycobacterial type III PKSs for long-chain to that these are the substrates in long-chain metabolites have not been isolated from that M. tuberculosis a rich of a remarkable array of long-chain and long-chain fatty J. 2002; PubMed Scopus Google Scholar). the M. tuberculosis genome of that are involved in the biosynthesis of substrates (1Cole S.T Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V Eiglmeier K. Gas S. Barry III C.E Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Barrell B.G Nature. 1998; 393: 537-544Crossref PubMed Scopus (6557) Google Scholar). modeling studies that the unusual to long-chain substrates might have evolved by the catalytic machinery of CHS and proteins. from a CHS active site an in has been along the in these mycobacterial proteins. modeling studies of PKS18 protein provide a Based on our structural we have investigated the importance of three acid of PKS18 The of to and in proteins that in structural of priming J.N. G. J. Chem. PubMed Scopus Google Scholar, H. S. J. C. 2002; 10: PubMed Scopus Google and our modeling studies that this in long-chain is that the to a has the in The and structural of in the are to A.M. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar). The CHS was investigated by to a The of this protein biosynthesis of catalytic with other CHS-like the of a acid the the number of J. J. Chem. PubMed Scopus Google Scholar, N. S. Biochem. J. 2002; PubMed Google Scholar). The the protein the starter the of using as a The protein was of studies have been for a from E. the protein not M. PubMed Scopus Google Scholar). We that this in the as a for acid Although is an in plant and type III is that are in the of that might the catalytic of The of an to the of the in has the of in that are the type III PKS superfamily Nat. Prod. Rep. PubMed Scopus Google Scholar, J. J. Chem. PubMed Scopus Google Scholar). In we have characterized two novel type III PKS proteins that the of using long-chain of the structural and mechanistic of thiolase enzymes have that type III PKS proteins might have by the of from homologous III proteins. The functional of mycobacterial type III PKSs to both CHS and III proteins fascinating the of the We K. for