Polyketide synthases (PKS) are enzyme complexes that synthesise many natural products of medicinal interest, notably a large number of antibiotics. The present work investigated the mupirocin biosynthesis system, comparing it with similar pathways such as thiomarinol and kalimantacin. The focus was on the structural modelling of the protein complexes involved in antibiotic synthesis, via molecular simulation and the analysis of structural and sequence data.
Structural docking of acyl carrier proteins (ACP) cognate for an HMG-CoA synthase orthologue responsible for β-methylation (MupH) identified key residues involved in the recognitions specificity of the interacting partners, further supported by mutagenesis experiments, which thus allows prediction of β-methylation sites in PKS. Moreover, complementation and mutagenesis experiments performed on MupH homologs from kalimantacin and thiomarinol systems suggests specificity between the ACP:HCS proteins in the β-branching suggesting the possibility of engineering multiple specific β-branching modifications into the same pathway.
Molecular dynamics simulations of ACPs from the mupirocin cluster revealed that the PKS ACPs form a cavity upon the attachment of the phosphopantetheine and acyl chains similar to what is seen in the fatty acid synthase ACPs and provide a better understanding of the structure function relationship in these small proteins. Molecular docking of the putative cognate substrate with the ketosynthase (KS) homo dimer of module 5 of the MmpA in the mupirocin pathway revealed a loop that may control specificity for the α-hydroxylated substrate and mutagenesis experiments support this proposition.
Paper(s) published on this work:
Farmer, R., Thomas, C.M., Winn, P.J., Modelling Polyketide Synthases and Similar Macromolecular Complexes, Current trends in Bioinformatics: an Insight, Chapter: 7, Wadhwa G, et al. (eds.), Springer Nature Singapore Pte Ltd., 2018, pp. 121-144. https://doi.org/10.1007/978-981-10-7483-7_7
Haines, A.S., Dong, X., Song, Z., Farmer, R., Williams, C., Hothersall, J., Płoskoń, E., Wattana-amorn, P., Stephens, E., Yamada, E., Gurney, R., Takebayashi, Y., Masschelein, J., Cox, R.J., Lavigne, R., Willis, C.L., Simpson, T.J., Crosby, J., Winn, P.J., Thomas, C.M., Crump, M.P., A conserved motif flags acyl carrier proteins for b-branching in polyketide synthesis, Nature Chemical Biology, 2013 Nov;9(11):685-92 [PMID: 24056399]
The work entitled above was conducted as part of my M.Tech. thesis during the period of Jan 2010 to July 2010. The project aimed to understand the Epigenetic changes due to histone modifications by a class of bacterial toxins. The gene expression data available in the public domain was used and the entire work was performed using various libraries and functions of Bioconductor software. My contribution in the work was to determine the genes that were differentially expressed due to histone modifications using statistical tests, followed by clustering analysis using various partition based and hierarchical based clustering methods. The final list of genes satisfying all the filtering criteria were annotated using Gene Ontology database. The results showed interesting findings that may contribute in understanding the effects of epigenetic changes in various host pathogen interactions and may also further our understanding about the mechanism of inheritance not depending on changes in the DNA sequence itself.
Project Title: Comparative Modelling of Steroidogenic Acute Regulatory Lipid Transfer Domains in Arabidopsis thaliana
Supervisor: Dr. Gitanjali Yadav
Steroidogenic Acute Regulatory Lipid Transfer (START) domains are responsible for transporting Cholesterol molecules from outer mitochondrial membrane to inner mitochondrial membrane for steroidogensis, usually found in mammals. However, recently they are also found in various plant genomes such as A. thaliana, Vitis vinifera, amongst others, in large number as compared to mammals. As the presence of Cholesterol in plants is still a debatable topic, finding of START domains may be hypothesized to be involved with plant signalling mechanisms by transporting various types of lipids. I was involved in this work during my B.Tech. final semester project from January 2008 to June 2008. I predicted theoretical model structures of all the 30 START domains in A. thaliana and carried out active site architecture analysis. The theoretical structure prediction was carried out by threading and comparative modelling methods, utilizing various online and standalone tools. Active site calculations were performed using VOIDOO and MAPMAN. The output files thus generated were analysed using self written Perl and Shell scripts. The results showed that START domains in plants vary in their active site volume and shape thus allowing a wide variety of molecules to bind however, the type of molecules that are likely to bind START domains are still need to be determined.
Paper published on this work:
Kumari, S., Shridhar, S., Singh, D., Farmer, R., Hundal, J., Priya, P., Sharma, P., Bhavishi, K., Schrick, K., and Yadav, G., The role of lectins and HD-ZIP transcription factors in Isoprenoid based plant stress responses, Proc. Ind. Natl. Sci. Acad, 2012; 78(4):671-691