Chemical biology of Mycobacterium tuberculosis: Deciphering the role of polyketide synthases in Mycobacteria

Project Associates/Assistants
Archana Vats
Uttara Marathe
R Krithika

Ph D Students
Omita Trivedi
Pooja Arora
Priti Saxena

Colloborators
Debasisa Mohanty
R Sankaranarayanan, CCMB, Hyderabad

Our laboratory has been interested in genome-based approaches to identify and exploit the microbial metabolic pathways that are involved in the biosynthesis of various natural products. The present focus is to understand the importance of various polyketide synthase gene clusters from Mycobacterium tuberculosis. The genome sequence of M.tuberculosis has revealed a remarkable array of genes that are homologous to polyketide synthases. Polyketide synthases (PKSs) are a class of enzymes that are involved in the biosynthesis of secondary metabolites such as erythromycin, rapamycin, tetracycline, lovastatin, and resveratrol. Our study attempts to understand and exploit the role of polyketide synthases in the biology of M.tuberculosis. The objectives are (i) identification and biochemical analyses of enzymes that are involved in the biosynthesis of metabolites, (ii) isolation and characterization of PKS gene products by heterologous expression of these genes in Streptomyces coelicolor and Escherichia coli, (iii) characterization of molecular mechanisms mediating the crosstalk between various polyketide synthases (PKSs) and fatty acid synthases (FASs) in M.tuberculosis, and (iv) genetic and/or chemical knock-out of PKS genes to synthesize novel polyketides and to study the effects of these changes on mycobacterial pathogenecity.

Characterization of Mycobacterial Type III PKSs

Three mycobacterial type III PKSs showed 40-45% similarity with both plant and bacterial CHS-like sequences. The expression of PKS18 protein was modulated to soluble form by inducing cultures with low IPTG concentration at 22°C. PKS11 protein could be purified in the soluble form by growing cultures without induction at 18°C for 24 hours. Despite our best efforts, PKS10 protein could not be expressed in soluble form. PKS18 and PKS11 proteins were purified by using Ni-NTA affinity chromatography and were then purified to homogeneity using anion exchange chromatography. Although our modeling studies predicted that mycobacterial proteins would not be able to use plant specific CoA-esters, we investigated the function of these proteins with several different CoA-esters due to their sequence similarity with plant PKSs. Several plant-specific CoA-esters were incubated with PKS18 and PKS11 proteins along with radiolabeled malonyl-CoA. Analysis of the reaction mixture by thin-layer chromatography (TLC) showed one faint radioactive band for PKS18 reactions with Rf value different from that of the expected plant products. Further examinations with longer time incubations clearly showed that PKS18 protein could synthesize this product from malonyl-CoA itself. Since PKS18 protein did not exhibit any activity with plant specific acyl-CoA substrates, we used several medium and long-chain fatty acid CoA-thioesters as starter substrates. It was noticed that several significant bands appear on the TLC plate, particularly for the long-chain acyl-CoA esters. Polyketide products biosynthesized from purified enzymes were separated on reverse phase HPLC and characterized by nanospray electrospray ionization mass spectrometry. Nanospray mass spectrometric analysis showed molecular ion peaks [M-H]- at m/z 265.17 and m/z 307.20, which could be expected respectively from triketide and tetraketide products of lauroyl-CoA reactions with extensions from malonyl-CoA. The starter unit specificity of PKS18 was investigated by performing steady state kinetic analysis using C2-C20 acyl-CoA substrates. Specificity of PKS18 proteins for long-chain acyl-CoA starters, as reflected in the KM values, is similar for C12 to C20 acyl chains. This unusual specificity towards long-chain aliphatic fatty acids is unparalleled in the chalcone synthase (CHS) family of condensing enzymes. The analysis of the structural models of PKS18 proteins clearly indicated that the putative CHS active site can not accommodate pyrone rings with long aliphatic side groups, suggesting the possibility of an altered binding pocket in these type III PKS proteins from M.tuberculosis. We have recently solved the crystal structure of this protein in collaboration with Dr Sankaranarayanan’s group at CCMB. Structural analysis has provided a testable model to probe into the functional relevance of catalytic and structural residues of mycobacterial PKSs.

Biochemical analyses of enzymatic crosstalk between FASs and PKSs

We are interested in examining the molecular basis of the enzymatic crosstalk between FASs and PKSs in mycobacteria through a combination of computational, genetic, biochemical and enzymological studies. The pps biosynthetic gene cluster, which is involved in the synthesis of complex lipid phthiocerol dimycocerosate (PDIM), has been used as a model system. This gene cluster spans approximately 50 kb of DNA and putatively codes for 16 ORFs. The pps cluster consists of (1) PKS genes (ppsA-E), (2) the FAS gene mycocerosic acid synthase (mas), (3) two ACS-like genes (fadD26 and fadD28, and (4) number of genes homologous to transporter proteins. The analyis of pps cluster have identified a number of unique features of modular PKS proteins. The PpsA protein contains an additional acyl carrier protein (ACP) domain located at the N-terminus end of the protein. The C-terminal end of ppsE protein possesses an unusual domain that is homologous to the condensation domain of non-ribosomal polypeptide synthetase (NRPS) proteins. Such domains have not been typically characterized in modular PKSs. We have also analyzed several other PKS-related proteins in M.tuberculosis including the FadD proteins. The dendrogram obtained from multiple sequence alignments of FadD proteins showed that these proteins formed two distinct clusters. A small cluster consisted of 12 FadD proteins with distinctly high sequence similarity, and showed homology to the adenylation domain of NRPS proteins. As a direct test to this hypothesis, we have functionally expressed and characterized number of FadD proteins in order to determine their importance to mycobacteria and understand their utility in different metabolic pathways. Biochemical reconstitution studies corroborated with our computational analysis, and confirmed the presence of two distinct groups of FadD proteins, one of which activate metabolic carboxylates as acyl-adenylates, while the others activate them as acyl-CoAs. The CoA-analogues are utilized by the host in lipid degradation as well as for providing precursors in the biosynthesis of metabolites. The acyl-AMP forming proteins have been shown to specifically transfer the activated fatty acids to the cognate PKSs, which are then further extended to produce complex lipids. Based on modular logic of modular PKSs, we have dissected the biosynthetic machinery of PDIM by dividing the complete process in four parts: 1). Activation and transfer of fatty acids on to PpsA protein, 2). Synthesis of phthiocerol by modular PKS proteins, 3). Synthesis and release of mycocerosic acids and 4). Transesterification of mycoserosic acids onto the phthiocerol. Cell-free reconstitution studies combined with mass spectrometric analysis has unambiguously identified functions of PKS and other associated proteins. Our studies thus reveal the mechanism involving biosynthesis of complex hybrid metabolites providing insights into how distinct biochemical functions are integrated within a given organism to generate metabolite diversity.

Publications

Original peer-reviewed articles

1.     *Yadav G, Gokhale RS and Mohanty D (2003) SEARCHPKS: A Program for detection and analyses of polyketide synthase domains. Nucl Acids Res 31:3654-3658 (*in press last year, since published).

2.     Saxena P, Yadav G, Mohanty D and Gokhale RS (2003) A new family of type III polyketide synthases in Mycobacterium tuberculosis. J Biol Chem 278:44780-44790.

3.     Trivedi OA., Arora P, Sridharan V, Tickoo R, Mohanty D and Gokhale RS (2004) Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature (in press).

4.     Ansari MZ, Yadav G, Gokhale RS and Mohanty D (2004) NRPS-PKS: A knowledge-based resource for analyses of NRPS/PKS megasynthases. Nucl Acids Res (in press).

5.     Rukmini R, Sanmugam VM, Saxena P, Gokhale RS and Sankaranarayanan R (2004) Crystallization and preliminary x-ray crystallographic investigations of an unusual type III polyketide synthase PKS18 from Mycobacterium tuberculosis. Acta Crystallogr D Biol Crystallogr (in press).

6.    Krishnamoorthy K, Gokhale RS, Contractor AQ and Kumar A (2004) Novel label-free DNA sensors based on poly (3,4-ethylenedioxythiophene). Chem Comm (in press).