Browsing by Author "Anand, Swadha"

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Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase
(PLOS, 2008) Chopra, Tarun; Banerjee, Srijita; Gupta, Sarika; Yadav, Gitanjali; Anand, Swadha; Surolia, Avadhesha; Roy, Rajendra P.; Mohanty, Debasisa; Gokhale, Rajesh S.
In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-b-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel ‘‘modularly iterative’’ mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.
Prediction of inter domain interactions in modular polyketide synthases by docking and correlated mutation analysis
(Taylor & Francis Group, 2013) Yadav, Gitanjali; Anand, Swadha; Mohanty, Debasisa
Polyketide synthases (PKSs) are huge multi-enzymatic protein complexes involved in the biosynthesis of one of the largest families of bioactive natural products, namely polyketides. The specificity of interactions between various catalytic domains of these megasynthases is one of the pivotal factors which control the precise order in which the extender units are joined during the biosynthetic process. Hence, understanding the molecular details of protein-protein interactions in the PKS megasynthases would be crucial for rational design of novel polyketides by domain swapping experiments involving engineered combinations of PKS catalytic domains. We have developed a computational method for exploring the binding interface between two proteins, and used it to identify the interacting residue pairs, which govern the specificity of recognition between acyl carrier protein (ACP) domain and two core catalytic domains, namely the ketosynthase (KS) and acyl transferase (AT). Both of these domain interactions i.e. the KS-ACP and the AT-ACP, are likely to play a major role in channelling of substrates and control of specificity during polyketide biosynthesis. The method, called interface scan, uses a combination of geometric docking and evolutionary information for the identification of the most appropriate mode of association between two proteins. The parameters of interface scan have been standardized based on analysis of contacts in the crystal structure of ACP in complex with ACP synthase (AcpS). Many of the contacts predicted for PKS domains are in agreement with available experiments.
SBSPKS: structure based sequence analysis of polyketide synthases
(Oxford University Press, 2010) Anand, Swadha; Prasad, M.V.R.; Yadav, Gitanjali; Kumar, Narendra; Shehara, Jyoti; Ansari, Md. Zeeshan; Mohanty, Debasisa
Polyketide synthases (PKSs) catalyze biosynthesis of a diverse family of pharmaceutically important secondary metabolites. Bioinformatics analysis of sequence and structural features of PKS proteins plays a crucial role in discovery of new natural products by genome mining, as well as in design of novel secondary metabolites by biosynthetic engineering. The availability of the crystal structures of various PKS catalytic and docking domains, and mammalian fatty acid synthase module prompted us to develop SBSPKS software which consists of three major components. Model_3D_PKS can be used for modeling, visualization and analysis of 3D structure of individual PKS catalytic domains, dimeric structures for complete PKS modules and prediction of substrate specificity. Dock_Dom_Anal identifies the key interacting residue pairs in inter-subunit interfaces based on alignment of inter-polypeptide linker sequences to the docking domain structure. In case of modular PKS with multiple open reading frames (ORFs), it can predict the cognate order of substrate channeling based on combinatorial evaluation of all possible interface contacts. NRPS-PKS provides user friendly tools for identifying various catalytic domains in the sequence of a Type I PKS protein and comparing them with experimentally characterized PKS/NRPS clusters cataloged in the backend databases of SBSPKS. SBSPKS is available at http://www.nii.ac.in/sbspks.html.