Technique Used for Study of Bio Synthetic Pathway

Basic Introduction to Biosynthetic pathway: Biosynthesis: Formation of a chemical compound by a living organism.

Biogenesis: Production or generation of living organisms from other living organisms.

 The living plants may be considered as a biosynthetic laboratory not

only for primary metabolites but also for a multitude of secondary metabolites.  Raw material used by plant for initiation of biosynthesis, water from soil and CO2 from atmosphere undergoes photosynthesis in presence of light.

6 CO2 + 6 H2O

C6H12O6 + 6O2

 Reaction involve in photosynthesis is: •

Light reaction or Hill reaction: utilize light energy

and concerned with the libration of oxygen and production of some reducing agent. • Dark reaction or Blackman reaction: utilize the energy from light reaction to fix CO2 into sugar.  Glucose is an intermediate product of photosynthesis and is the starting material from which complex food formed.  Complex form of glucose are starch , a portion of which converted into oils and a part is employed together with nitrogen, sulphur, phosphorus and other minerals form protein and complex organic compound.  Plant produce these compounds through various vital biosynthetic process: • Glycolysis and Krebs cycle helpful in production of secondary metabolites. • Pentose phosphate pathway for sugar. • Acetate-Mevalonate pathway for terpenoid and cardiac glycoside. • Shikimic acid pathway for cynogenetic glycoside, alkaloid and aromatic amino acid.

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 Metabolic product and intermediate form during biosynthesis is controlled by various enzymes.  Types of Metabolite :

Primary metabolite: primary metabolites are required for general growth and physiological activity of plants because of their basic metabolism. E.g. amino acid, fatty acid, nucleic acid, carbohydrate, protein. • Secondary metabolite: Secondary metabolites are derived biosynthetically from the primary metabolites but usually restricted to specific taxonomical group. They may represent chemical adaptations to environmental stresses or they may serves as defensive or protective against microorganism, insect and higher herbivorous predator. E.g. alkaloid, glycoside, volatile oil, flavonoid, lignan, carotenoid etc.  Other reaction involve to complete biosynthesis: •

• • • • • •

Oxidation Reduction condensation Amination Methylation Cyclization etc.

Precursor for Alkaloid biosynthesis:  Lysine: Piperidine and Quinazoline

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Ornithine: pyrrolizidine Tyrosine: isoquinoline Tryptophan: Indole Phenyl alanine: Ephedrine, mescaline Dihydroxyphenyl alanine: Emetine and Colchicine

ORIGIN OF SOME SECONDARY METABOLITE

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TECHANIQUES USED FOR INVESTIGATION OF BIOSYNTHETIC PATHWAYS     

Tracer techniques Use of Isolated organs or Tissues Grafting method Use of mutant strains Enzymatic studies

1. TRACER TECHANIQUES: o A technique which utilizes a labeled compound to trace or find out the

different intermediates and various steps involved in biosynthetic process in plant at given rate and given time. o When these labeled compounds are administered into the plants, they become a part of general metabolic pool and undergo reactions characteristic to the metabolism of that particular plant. SIGNIFICANCE OF TRACER TECHANIQUES o Tracing of biosynthetic pathway by incorporating radioactive

isotopes into the precursor or starting material. E.g. By incorporation of C14 to phenylalanine, the biosynthesis of cynogenetic glycoside, prunacin can be traced. o Location and quantity of the compound can be determined in biological system. o Different trace element for different studies:  For studies on Protein, alkaloid and amino acid: nitrogen atom gives more specific information than carbon atom.  For studies on glycosidic linkage: O, N, S, and C atom.  For studies on Terpenoids: O-atom BASIC STEPS INVOLVED IN TRACER TECHANIQUES:

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Preparation of labeled compound Incorporation of labeled compound to tissue system Separation or isolation of labeled compound from tissue system Determination of nature of metabolites in various biochemical fraction

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PREPARATION OF LABELED COMPOUND: The labeled compounds may be prepared by use of radioactive isotopes and stable isotopes. Radioactive isotopes: C14, H3, S35, P32, I131,Co60 Stable isotopes: H2, C13, N15, O18 Properties of some radioactive isotopes: Natural C12 H1 S32 P31 Cl35 I127 Co59

Radioactive C14 H3 S35 P32 Cl36 I131 Co60

Radiation beta beta beta beta beta Beta, Gamma Beta, Gamma

Half life 5760 yrs 12.5 yrs 871 days 14.3 days 4.4 × 105yrs 8 days 5.3 days

Criteria for selection of trace element: • The starting concentration of trace element must be sufficient enough to withstand dilution in the course of metabolism • For proper labeling the physical and chemical nature of the compound must be known. • Half life of the tracer isotope should be sufficiently long. • The labeled compound should not be damage the Tissue system In biological investigations, the use of radioactive isotopes enables the metabolism of compounds to be followed in living organism for detection and estimation of soft and easily absorbed radiation from labeled compound. The instrument of choice to detect the properties of metabolite is scintillation counter, GM-counter, Ionization chamber, Mass spectroscopy, NMR etc. E.g. growing chlorella in an atmosphere containing 14CO2.

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 Tritium labeling is effected by catalytic exchange (Pt catalyst) in aqueous media by hydrogenation of unsaturated compounds with tritium gas. INTRODUCTION OF LABELED COMPOUND TO TISSUE SYSTEM: There are six methods use to incorporate labeled compound to tissue system:  Root feeding  Stem feeding  Direct injection  Infiltration  Floating method  Spraying technique ROOT FEEDING: The plant in which roots are the biosynthetic sites, this method is preferred. E.g.Tobacco In this type of experiment the plant are cultivated hydroponically to avoid microbial contamination. STEM FEEDING: Presence of root don’t require for biosynthesis. In this method substrate can be administered through the cut ends of stem immersed in a solution. For Latex containing plant this method is not suitable. DIRECT IJECTION: This method is applicable to the plant with hollow stem. E.g. Umbelliferae and capsule bearing plant (opium poppy) INFILTERATION: Wick feeding When it is desired to carryout feeding on plant rooted in soil or other support without disturbing the root, wick feeding is applicable. FLOATING METHOD:

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When small amount of material is available, floating method is used. In this method, leaf disc or chopped leaves are floating on the substrate solution. This technique is also used in conjugation with vacuum infiltration to remove gases. SPRAY TECHNIQUE: In this method compound have been absorbed after being sprayed on leaves in aqueous solution. E.g. Steroids. SEPARATION OR ISOLATION OF LABELED COMPOUND OR METABOLITE: Depending on the nature of drug and its source different method of extraction is employed.  Soft and fresh tissue: Infusion, maceration  Hard tissue: Decoction and Hot percolation  Unorganized drug: Maceration with adjustment Choice of solvent for extraction:  Fat and oil: non polar solvent  Alkaloid, Glycoside, Flavonoid: slightly polar solvent  Plant phenol: Polar solvent • Fractional crystallization, Partition, column chromatography also used as separation technique. DETERMINATION OF NATURE OF METABOLITES: Depending on nature of isotopes various instrumentation techniques is used for determination of chemical nature of intermediate and final product. For radioactive isotopes, • GM-counter • Scintillation or liquid scintillation counter • Ionization chamber

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These entire instruments characterize the nature of radiation. Basically it depends upon the conversion of kinetic energy of particle into fleeting pulse of light as a result of its penetration into a suitable luminescent medium. For stable isotopes, • Mass spectroscopy- gives molecular peaks depending on mass/charge ratio. • NMR- gives nature of carbon and proton. Methods in Tracer Techniques Precursor- Product Sequence: • For elucidation of biosynthetic pathways in plants by means of labeled compounds, the precursor product sequence method is used. • In this method presumed precursor of the constituent is under investigation in a labeled form is fed to plant and after a particular time the constituent is isolated, purified and its radioactivity is determined. • This method is extensively applied to the biogenesis of morphine and ergot alkaloid Competitive feeding: This method is normally used to determine two possible intermediates in the plants. B A

C

B1 Competitive feeding can distinguish whether B or B1 is the normal intermediate in the formation of C from A. E.g. Biosynthesis of Hemlock alkaloids (Coniine and conhydrine) Sequential Analysis: The principle of this method of investigation is to grow plants in the atmosphere of 14CO2 and by analysis of plants at a given time intervals, to

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obtained the sequence in which various related compounds becomes labeled from the results some biosynthetic route may be accepted and others rejected. • 14CO2 and sequential analysis has been very successfully used in the elucidation of the path of carbon in photosynthesis. • Determination of sequential formation of opium and tobacco alkaloids. 2. USE OF ISOLATED CELL, TISSUE AND ORGAN: • Isolated cells, tissues and organ grown on nutrient medium aseptically are being utilized for the elucidation of biosynthetic pathways of secondary metabolites. • The plant tissue culture technique affords considerable potential in investigation of biogenetic pathways as the material obtained is uniform in every respect and is also available at all time. • This cultured material is easily manageable and reproducible and easier to feed with potential precursor of the secondary metabolites under study. • For analysis of products sample to be taken repeatedly and effects of microbes on the precursor or its product is eliminated. 3. ENZYMATIC STUDIES: Enzymatic studies involving the enzymes catalyzing the metabolic reactions, which are also helpful in the investigation of biosynthetic pathways. Enzymes can be isolated and studied. Two isomers of chorismate mutase enzyme have been isolated from poppy seedling and characterize. 4. GRAFTING METHOD: Grafted plant is especially use in finding the main site of primary as well as secondary metabolite formation. Grafting techniques have been useful in providing principle sites:  For capsicin in developing capsicum plants  Aerial plants parts of cannabis for resin  Roots of nicotiana tobacum for nicotine  Leaves ofnicotiana glauca for anabasine

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5. Mutant strand: By selecting mutant strand, biosynthetic pathways studies are possible. To produce a mutant strand physical or chemical methods are employed. Physical: Radiation Chemical: Colchicin.

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