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Andrographis paniculata (Burm.f.) Wall. ex Nees (Ap) contains andrographolide, i.e., synthesized via cytosolic MVA and plastidial MEP pathways using IPP and DMAPP as precursor molecules. HMGR catalyzes the first committed and rate-limiting step in MVA pathway. Limited information is available on structural and functional aspects of key enzyme HMGR. In a preliminary study, the in vivo leaves and light/dark grown callus was used for protein profile analysis. The callus growth and andrographolide content through HPLC analysis was carried out. SDS-PAGE total protein profile of in vivo leaves, light, and dark grown callus was investigated. A high intensity band of 62.3 KD protein indicating its proximity with HMGR enzyme was observed in leaves and light callus compared to dark grown callus. On this background, the present communication reports bioinformatics analysis of HMGR enzyme to decipher its functional and structural properties. The isoelectric point (pI), molecular weight and hydrophobicity were calculated using Expasy tools. The secondary (2D) and tertiary (3D) structures were predicted and validated using PROCHECK SAVES algorithm. Our finding on the allowed percentage (88.2%) of amino acid residues in the Ramachandran plot indicated that the simulated 3D structure was reliable. Phylogenetic analysis of A. paniculata HMGR (ApHMGR) with different plants and other organisms including humans revealed close relationships and common lineage. The protein-protein interaction studies using STRING 10 tool revealed a close association of HMGR and nine other proteins in MVA and MEP pathways. The present analyzed data will give an insight on the structural and function attributes corresponding to this important HMGR protein. The molecular and biochemical understanding of HMGR protein will be advantageous for andrographolide yield enhancement through metabolic engineering.

Andrographis paniculata (Burm.f.) Wall. ex Nees is as an important medicinal plant from centuries for treating infectious diseases in India and other countries. Active principles of this plant are diterpene lactones, specifically andrographolides and are synthesised via two independent biosynthetic pathways, i.e., mevalonic acid (MVA) and methyl-erythritol phosphate (MEP) pathway. Very meagre genomic and proteomic information is available about the genes and enzymes involved in these biosynthetic pathways. In this study, we have performed in silico characterization of a vital rate limiting enzyme 1-deoxy-D-xylulose 5phospahte synthase (DXS) of MEP pathway in A. paniculata. Structural and functional characterization of A. paniculata DXS (ApDXS) revealed its hydrophobic nature and a non trans-membrane protein was found to be present in chloroplast stroma. Predicted 3D structure with phyre2 tool had shown 85% of amino acids in the most favoured region as revealed by Ramachandran plot and was 96.32% structurally reliable. The phylogenetic analysis of ApDXS by MEGA7 revealed evolutionarily close relationship with Lamiaceae (Phlomis umbrosa/ Phlomoides umbrosa, Plectranthus barbatus, Lavandula angustifolia and Salvia miltiorrhiza) and Pedaliaceae (Sesamum indicum) families. Protein-protein interaction study revealed the interface of ApDXS with other MEP pathway proteins such as HDS, HDR, DXR and CDPMEK. Further, interaction was also evident with MVA pathway protein HMGS and downstream proteins, viz., GPS1, IPPI1 of diterpenoid pathway. The findings on the interactions of ApDXS with HMGS has given insight to the cross talk between MEP and MVA pathways. Prediction of bio-physico-chemical properties, secondary and tertiary structures will be of significance in protein purification processes. In addition, it will also be advantageous for drug designing applications with particular reference to manipulation of biosynthetic pathway, involving diterpene lactones in A. paniculata.