Prashant Sonawane

Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala

Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.

Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1).

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants. Abbreviations Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.

Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies

Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens. Abbreviations IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.

Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2.

Conformational transitions of cinnamoyl CoA reductase 1 from Leucaena leucocephala.

Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2M GdnHCl (as compared to drastic loss of native structure in 2M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein.

Genetic Engineering of Phenylpropanoid Pathway in Leucaena leucocephala

The phenylpropanoid pathway is responsible for the biosynthesis of a variety of products that include lignin, flavonoids and hydroxycinnamic acid conjugates. Many intermediates and end products of this pathway play important role in plants as phytoalexins, antiherbivory compounds, antioxidants, ultra-violet (UV) protectants, pigments and aroma compounds. Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism a globally important part of plant biochemistry. The mechanical support provided by lignin prevents lodging, a problem in many agronomically important plants, it also provides a hydrophobic surface, essential for longitudinal water transport, and provides a barrier against pathogens. Finally the many functions of lignin and related products in resistance to biotic and abiotic stresses make the phenylpropanoid pathway vital to the health and survival of plants. Besides its critical role in normal plant health and development, high lignin levels are problematic in the agro-industrial exploitation of various plant species. Lignin is considered an undesirable component in paper manufacture, and has a negative impact on forage crop digestibility. Leucaena leucocephala is one of the most versatile, fast growing commercially important trees for paper and pulp industry in India, contributing 1/4th of the total raw material. Lignin composition, quantity and distribution are known to affect the agro-industrial utilization of plant biomass. High quantity and low Syringyl (S) to Guaiacyl (G) lignin ratio plays a detrimental role in economy and ecology of paper production. Every unit increase in S/G ratio decreases the cost of paper production by two and half times. Moreover chemical processing of pulp for lignin removal releases chlorinated organic compounds in effluent, which are hazardous and a serious threat to the environment. Hence, there is currently intense interest in modifying the content and/or composition of the cell wall structural polymer (lignin) as a means of improving the efficiency of the paper pulping process for forest trees. To engineer plants with agronomically useful lignin related traits, we need to devise strategies that can flexibly and predictably yield reductions in lignin content and/or changes in lignin monomer composition. Our studies have concentrated on attempts to alter the levels of enzymes involved in early as well as late phenylpropanoid pathway, mainly by downregulation or upregulation of the phenylpropanoid pathway genes in transgenic L. leucocephala and tobacco plants. Besides, we are also working on some R2R3 type MYB transcription factors supposed to play important role in lignin biosynthesis and some other genes which are not directly involved in phenylpropanoid pathway, but are important for carrying out polymerization of monolignols (peroxidases) and defense mechanism of plants (β glucosidase: family 1 Glycosyl hydrolase). Major phenylpropanoid pathway genes (C4H, 4-CL, CCoAOMT, CCR, Cald5H and CAD) were isolated using PCR based approach. Their 5’ and 3’ UTR determined by rapid amplification of cDNA race (RACE). We could isolate multiple isoforms of most of the genes in this way well supported by Southern hybridization experiment. All the genes were expressed in E. coli and/or yeast with/without the signal sequence. The over-expressed proteins were purified using suitable methods and were used for raising polyclonal antibodies against them. The most un-conserved region of each gene was cloned in antisense orientation in suitable binary vector and L. leucocephala and tobacco explants were transformed using the antisense construct to down-regulate the targeted gene. In order to increase the S/G ratio of lignin monomers sense construct of the target gene was used. We have also done RNAi based downregulation of Cinnamate 4-Hydroxylase (C4H), a key enzyme of phenylpropanoid pathway and a member of cytochrome P450 family in tobacco. Spatio-temporal expression of each gene was studied in L. leucocephala in different tissues at different periods of their growth. Kinetics study of some of the enzymes has been carried out in our lab. We are also trying to establish the structure function correlation of some enzymes. We have also isolated promoters of some phenylpropanoid pathway genes and have identified R2-R3 type MYB binding domain(s) in them. Two MYB genes have been isolated from L. leucocephala and heterologously expressed in Escherichia coli. Their DNA binding efficiency and role in regulating phenylpropanoid pathway remain to be seen. All the isolated genes shared 70-80% homology with the already reported sequences from other species at nucleotide level and more than 80% identity at amino acid level. Different isoforms of different genes had varying degree of identity between them ranging from 80% to more than 95%. We could easily locate the ribosome binding site in the 5’ UTR and the polyadenylation signal in the 3’ UTR in all the genes. Proteins were expressed in both prokaryotic and eukaryotic system. Some of the genes were difficult to express in BL21 (DE3), primarily because of the translational incompatibility of some of the codons in E. coli and may be partly because of the signal sequence present in most of the proteins. All the genes were found to be actively expressing in lignifying tissues and roots in comparison to leaves albeit a time dependent regular expression pattern could not be drawn in case of every gene. We used three methods of genetic transformation for transferring our gene constructs to L. leucocephala embryo viz: Agrobacterium mediatd, gene gun mediated and gene gun followed by cocultivation with Agrobacterium. The transformation and regeneration efficiency varied with each protocol. The transgenic plants invariably showed stunted vigour and slow growth irrespective of the nature of the gene downregulated or upregulated. Initial screening of the transformants was done on MS medium containing appropriate antibiotic and later confirmation was done using PCR with hptII/nptII, gus specific and CaMV35S promoter specific primers. At least one tobacco plant downregulated for C4H showed rudimentary root system and curled leaves with brown tip. Transformed Leucaena and tobacco plants had reduced lignin content with varying degree in case of every downregulated gene. Histochemical staining of transverse root and stem tissue sections showed reduced lignification as evidenced by immunocytolocalization patterns of the candidate protein under study. Promoters of few lignin biosynthetic pathway genes having R2R3 type MYB-binding signal sequences (AC elements) have been cloned. Two R2R3 type MYB transcription factors from L. leucocephala that have been expressed and purified from E. coli will be used for gel retardation studies with the promoter sequences. Also in vitro synthesized oligonucleotides having the highly conserved MYB-binding motifs will be designed to determine the most probable binding sites of the two MYB proteins. Sense construct for the two MYB genes have been transformed in tobacco and antisense construct have been transformed in Leucaena plants to study the after effects of gene manipulation. In short, we have isolated and characterized several genes belonging to phenylpropanoid pathway and have expressed them in different systems. Transgenics for down-regulation or upregulation of those genes have shown very interesting results.

Enhanced activity of Withania somnifera family-1 glycosyltransferase (UGT73A16) via mutagenesis

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein–ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2–3 fold and 6–7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.