Polyphenol oxidase (PPO) arm of catecholamine pathway catalyzes the conversion of L-tyrosine to L-DOPA in Mucuna pruriens (L.) DC var. pruriens: An integrated pathway analysis using field grown and in vitro callus cultures.

Mucuna pruriens (L.) DC var. pruriens is the natural source for L-DOPA, precursor of the neurotransmitter dopamine, used widely in the treatment of Parkinson's disease. However, L-DOPA synthesis in plants is mediated either by Catecholamine (CA) pathway or alternate pathway catalyzed by Cytochrome P450 (CYP450) class of enzymes. Interestingly, the CA pathway itself can be initiated either by tyrosine hydroxylase (TH) or polyphenol oxidase (PPO). The CA pathway mediated synthesis of L-DOPA has not yet been proved in M. pruriens albeit strong indications. Therefore, the present investigation is focused on metabolite analysis of major intermediates of CA pathway up to the formation of dopamine and expression analysis of the selected genes, in different tissues and callus cultures. The four major intermediates, L-tyrosine, tyramine, L-DOPA and dopamine, were detected using NMR spectroscopy and quantified by HPLC in the callus cultures and in different tissues of the field plant, respectively. The various stages of leaf tissue were also analyzed for metabolite profiling. The relative amount of intermediates detected during the ontogeny of leaf indicates that PPO mediated conversion of L-tyrosine to dopamine through L-DOPA is relatively higher compared to dopamine production from tyramine. Among the two possible enzymes, activity of PPO was 6.5-fold more than TH in metabolically active young leaves compared to intermediate leaves. The gene expression profiles comprising upstream genes of L-tyrosine synthesis and downstream up to dopamine synthesis shows strong correlation with L-DOPA synthesis. The study validates CA pathway mediated synthesis of L-DOPA with PPO as candidate enzyme, in M. pruriens.

Hypericin biosynthesis in Hypericum hookerianum Wight and Arn: investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization.

The biochemical pathway to hypericin biosynthesis is presumed to be polyketide synthase (PKS) mediated, but it has not been experimentally validated, and no alternate route (chorismate/o-succinylbenzoate pathway) has been analyzed. We report here our earlier developed auxin inducible culture systems of Hypericum hookerianum as a model, to study the metabolic pathway to hypericin synthesis. Inhibitors of the alternate pathway at varying concentrations showed steady synthesis of total hypericins with means of 2.80±0.22, 18.75±0.01; 16.39±3.75, 29.60±1.90 (mevinolin) 2.53±0.10, 18.12±0.56; 0.14±0.01, 14.28±1.11 (fosmidomycin) and 2.7±0.35, 18.75±0.61; 0.14±0.01, 12.80±1.09 mg g(-1) DW (glyphosate) in the control and auxin-induced shoot and shoot-forming callus cultures, respectively. SSH analysis classified the differentially expressed sequences into protein synthesis (38%), modification (20%), electron transport (9%) and remaining as unclassified (11%) and unknown proteins (22%). Functional annotation of sequences indicates the presence of additional protein components besides PKS activity. Our results demonstrate direct biochemical and molecular evidence of PKS hypothesis of hypericin biosynthesis for the first time.