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1.  Novel autoproteolytic and DNA-damage sensing components in the bacterial SOS response and oxidized methylcytosine-induced eukaryotic DNA demethylation systems 
Biology Direct  2013;8:20.
The bacterial SOS response is an elaborate program for DNA repair, cell cycle regulation and adaptive mutagenesis under stress conditions. Using sensitive sequence and structure analysis, combined with contextual information derived from comparative genomics and domain architectures, we identify two novel domain superfamilies in the SOS response system. We present evidence that one of these, the SOS response associated peptidase (SRAP; Pfam: DUF159) is a novel thiol autopeptidase. Given the involvement of other autopeptidases, such as LexA and UmuD, in the SOS response, this finding suggests that multiple structurally unrelated peptidases have been recruited to this process. The second of these, the ImuB-C superfamily, is linked to the Y-family DNA polymerase-related domain in ImuB, and also occurs as a standalone protein. We present evidence using gene neighborhood analysis that both these domains function with different mutagenic polymerases in bacteria, such as Pol IV (DinB), Pol V (UmuCD) and ImuA-ImuB-DnaE2 and also other repair systems, which either deploy Ku and an ATP-dependent ligase or a SplB-like radical SAM photolyase. We suggest that the SRAP superfamily domain functions as a DNA-associated autoproteolytic switch that recruits diverse repair enzymes upon DNA damage, whereas the ImuB-C domain performs a similar function albeit in a non-catalytic fashion. We propose that C3Orf37, the eukaryotic member of the SRAP superfamily, which has been recently shown to specifically bind DNA with 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, is a sensor for these oxidized bases generated by the TET enzymes from methylcytosine. Hence, its autoproteolytic activity might help it act as a switch that recruits DNA repair enzymes to remove these oxidized methylcytosine species as part of the DNA demethylation pathway downstream of the TET enzymes.
This article was reviewed by RDS, RF and GJ.
PMCID: PMC3765255  PMID: 23945014
2.  Modeling holo-ACP:DH and holo-ACP:KR complexes of modular polyketide synthases: a docking and molecular dynamics study 
Modular polyketide synthases are multifunctional megasynthases which biosynthesize a variety of secondary metabolites using various combinations of dehydratase (DH), ketoreductase (KR) and enoyl-reductase (ER) domains. During the catalysis of various reductive steps these domains act on a substrate moiety which is covalently attached to the phosphopantetheine (P-pant) group of the holo-Acyl Carrier Protein (holo-ACP) domain, thus necessitating the formation of holo-ACP:DH and holo-ACP:KR complexes. Even though three dimensional structures are available for DH, KR and ACP domains, no structures are available for DH or KR domains in complex with ACP or substrate moieties. Since Ser of holo-ACP is covalently attached to a large phosphopantetheine group, obtaining complexes involving holo-ACP by standard protein-protein docking has been a difficult task.
We have modeled the holo-ACP:DH and holo-ACP:KR complexes for identifying specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. A novel combination of protein-protein and protein-ligand docking has been used to first model complexes involving apo-ACP and then dock the phosphopantetheine and substrate moieties using covalent connectivity between ACP, phosphopantetheine and substrate moiety as constraints. The holo-ACP:DH and holo-ACP:KR complexes obtained from docking have been further refined by restraint free explicit solvent MD simulations to incorporate effects of ligand and receptor flexibilities. The results from 50 ns MD simulations reveal that substrate enters into a deep tunnel in DH domain while in case of KR domain the substrate binds a shallow surface exposed cavity. Interestingly, in case of DH domain the predicted binding site overlapped with the binding site in the inhibitor bound crystal structure of FabZ, the DH domain from E.Coli FAS. In case of KR domain, the substrate binding site identified by our simulations was in proximity of the known stereo-specificity determining residues.
We have modeled the holo-ACP:DH and holo-ACP:KR complexes and identified the specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. Analysis of the conservation profile of binding pocket residues in homologous sequences of DH and KR domains indicated that, these results can also be extrapolated to reductive domains of other modular PKS clusters.
PMCID: PMC3422181  PMID: 22639887
Molecular dynamics; Protein-ligand docking; Protein-protein interaction; Substrate binding site; Evolutionary conservation; Modular polyketide synthase; Dehydratase domain; Ketoreductase domain
3.  SBSPKS: structure based sequence analysis of polyketide synthases 
Nucleic Acids Research  2010;38(Web Server issue):W487-W496.
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
PMCID: PMC2896141  PMID: 20444870
4.  Novel Intermolecular Iterative Mechanism for Biosynthesis of Mycoketide Catalyzed by a Bimodular Polyketide Synthase  
PLoS Biology  2008;6(7):e163.
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-β-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.
Author Summary
Polyketide synthases (PKSs) form a large family of multifunctional proteins involved in the biosynthesis of diverse classes of natural products. Mycobacterium tuberculosis (Mtb) exploits these polyketide biosynthetic enzymes to synthesize complex lipids, many of which are essential for its virulence. PKSs utilize two common mechanistic themes to produce these metabolites: (1) modular—in which each set of catalytic sites is used only once during the entire biosynthetic process and (2) iterative—in which the same set of active sites is used repeatedly. Our study with PKS12 protein from Mtb (the largest protein in this genome) reveals a third mechanism for polyketide biosynthesis. In this hybrid “modularly iterative” mechanism, PKS12 protein forms a supramolecular assembly to perform repetitive cycles of iterations. The protein assembly is formed by specific intermolecular interactions between N- and C-terminus linkers, analogous to modular PKSs. Our study adds a new dimension to the existing catalytic and mechanistic versatility of PKSs, providing a new perspective on how metabolic diversity could be generated by different combinations of existing functional scaffolds.
A novel iterative biosynthetic mechanism for multifunctional polyketide synthases reveals how the metabolic diversity of this enzyme family can arise by using existing scaffolds in novel combinations.
PMCID: PMC2443190  PMID: 18613748

Results 1-4 (4)