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 ␣-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 smallchain precursors. The specificity of PKS18 and PKS11 proteins toward long-chain aliphatic acyl-coenzyme A (C 12 to C 20 ) 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 ␤-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 CHSlike 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 (1, 2). 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). 1 Although a polyketide producthas not yet been isolated from M. tuberculosis, recent studies have implicated some of its PKS genes in the biosynthesis of complex lipids (3). In this report, we have characterized two novel polyketide synthases from M. tuberculosis, which are involved in the biosynthesis of unusual long-chain ␣-pyrones.Polyketides are a diverse class of secondary metabolites that possess a broad range of biological activities (4). 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 reaction...

Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis

“…This makes the carbonyl function the most likely target of the reduction reaction that affects only the tetraketide compounds. Moreover, in collision-induced dissociation experiments ( Figures 7B to 7D), we observed the presence of fragments at the 125 m/z value in the mass spectrum of reduction reaction products ( Figures 7C and 7D) and in the tetraketide substrate ( Figure 7B), which is diagnostic for the lactone ring of the tetraketide a-pyrones (Saxena et al, 2003;Rubin-Pitel et al, 2008). Thus, these results suggest that the lactone ring remained unaffected by the reduction reaction.…”

Type III polyketide synthases in natural product biosynthesis

“…The physiological role of this operon remains unknown, as deletion of these two genes did not cause any detectable phenotypic changes (36). Finally, PKS18 from Mycobacterium tuberculosis has been shown to accept longchain aliphatic acid CoA-esters (C 6 -C 20 ) as the starter units to produce triketide or tetraketide pyrones via two or three condensation reactions (37). The crystal structure of PKS18 reveals a 20 Å substrate binding tunnel that allows this synthase to utilize a broad range of substrates, similar to PhlD (30).…”

“…The crystal structure of PKS18 reveals a 20 Å substrate binding tunnel that allows this synthase to utilize a broad range of substrates, similar to PhlD (30). In addition to PKS18, several bacterial type III PKSs including PKS11 and SCO7671 from S. coelicolor were found to use long-chain fatty acid CoA-esters as the starter units (37).…”

Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies

“…Moreover, these same mutants constructed in strains other than H37Rv retained the ability to synthesize PDIM (authors' unpublished observations). Indeed, there are even a few recorded instances in the literature where independent laboratories have published discordant results with respect to the proposed involvement of particular genes in PDIM biosynthesis (Matsunaga et al, 2004;Saxena et al, 2003;Sirakova et al, 2003;Waddell et al, 2005). In hindsight, it seems likely that the loss of PDIM in these instances was unrelated to the pathways being studied.…”