Metabolic pathway including primary and secondary pathway; Shikimic acid, mevalonic acid and Amino acid pathway biogenetic investigation :isolated organ, grafting method, tracer techniques

1. Metabolic pathways in higher plants and
their determination
A. Brief study of basic metabolic pathways and formation of different
secondary metabolites through these pathways- Shikimic acid
pathway, Acetate pathways and Amino acid pathway.
B. Study of utilization of radioactive isotopes in the investigation of
Biogenetic studies.

2. Contents
 Introduction to metabolites
 Primary & secondary metabolite difference
 Metabolic pathway of Primary and secondary metabolites
 Shikimic acid pathway
 Acetate pathways
 Amino acid pathway.

3. Introduction
 Biosynthesis is a process of forming larger organic compounds from
small subunits within a living organism.
 Metabolites are the organic compound synthesised by plants using
enzyme mediated chemical reactions through the pathway known
as Metabolic pathway.
 These metabolites are of two types i.e., Primary metabolites and
Secondary metabolites.

4. Primary metabolites
 These are synthesized through
primary metabolic pathway.
 Molecules that are essential for
growth and development of an
organism.
 Therapeutically inactive
 Examples:
Carbohydrates ,Proteins , Lipids,
Nucleic acids , Hormones
Secondary metabolites
 These are synthesised through
secondary metabolic pathway
 Molecules that are not essential
for growth and development of
an organism.
 Therapeutically active
 Examples:
Alkaloids, Glycosides , Terprnoids

6. SHIKKIMIC ACID PATHWAY
 The Shikimic acid pathway is a key intermediate from carbohydrate for the
biosynthesis of C6-C3 units (phenyl propane derivative).
 The Shikimic acid pathway converts simple carbohydrate precursors
derived from glycolysis and the pentose phosphate pathway to the
aromatic amino acids.
 The shikimate pathway is a 7 step metabolic route used by bacteria,
fungi, Algae, parasites, and plants for the biosynthesis of aromatic amino
acids (phenylalanine, tyrosine, and tryptophan).
 This pathway is not found in animals; therefore, phenylalanine and
tryptophan represent essential amino acids that must be obtained from the
animal's diet.
 Animals can synthesize tyrosine from phenylalanine, and therefore is not an
essential amino acid except for individuals unable to hydroxylate
phenylalanine to tyrosine).

8.  Phosphoenolpyruvate and erythrose-4-phosphate react to form 2-keto3- deoxy7
phosphoglucoheptonic acid, in a reaction catalyzed by the enzyme DAHP
synthase.
 2-keto-3-deoxy-7-phosphoglucoheptonic acid is then transformed to 3-
dehydroquinate (DHQ), in a reaction catalyzed by DHQ synthase.
 Although this reaction requires nicotinamide adenine dinucleotide (NAD) as a
cofactor, the enzymic mechanism regenerates it, resulting in the net use of no NAD.
 DHQ is dehydrated to 3-dehydroshikimic acid by the enzyme 3-dehydroquinate
dehydratase, which is reduced to shikimic acid by the enzyme shikimate
dehydrogenase, which uses nicotinamide adenine dinucleotide phosphate
(NADPH) as a cofactor.
 The next enzyme involved is shikimate kinase, an enzyme that catalyzes the ATP
dependent phosphorylation of shikimate to form shikimate 3-phosphate.
 Shikimate 3-phosphate is then coupled with phosphoenol pyruvate to give 5-
enolpyruvylshikimate-3-phosphate via the enzyme 5-enolpyruvylshikimate-3-
phosphate (EPSP) synthase.
 Then 5-enolpyruvylshikimate-3-phosphate is transformed into chorismate by a
chorismate synthase.

9. ROLE OF SHIKKIMIC ACID PATHWAY
 Starting Point in The Biosynthesis of Some Phenolics Phenyl alanine and
tyrosine are the precursors used in the biosynthesis of phenylpropanoids.
 The phenylpropanoids are then used to produce the flavonoids, coumarins,
tannins and lignin.
 Gallic acid is formed from 3-dehydroshikimate by the action of the enzyme
shikimate dehydrogenase to produce 3,5- didehydroshikimate.
 The latter compound spontaneously rearranges to gallic acid.
Other compounds
Shikimic acid is a precursor for:
 Indole, indole derivatives and aromatic amino acid tryptophan and
tryptophan derivatives & many alkaloids and other aromatic metabolites.

11. MEVALONIC ACID PATHWAY
 The Acetate mevalonate pathway, also known as the isoprenoid pathway
or HMG-CoA reductase pathway is an essential metabolic pathway present
in eukaryotes, archaea, and some bacteria.
 Acetate occupies central position in relation to general metabolism, it
gives straight chain compounds as well as aromatic compounds viz.,
Terpenoids, steroids and fatty acids.
 The mevalonate pathway begins with acetyl-CoA which is an active form
of Acetate
 Acetate pathway has two main routes
A. Acetate mevalonate pathway/Isoprenoid pathway -> Terpenes & steroids
B. Acetate melonate pathway -> Fatty acid & lipids

12.  Mevalonic acid pathway or isoprenoid pathway biosynthesises active
building block i.e., Isoprene unit (C5 ) for all the isoprenoid compounds .
 The pathway produces two five-carbon building blocks called isopentenyl
pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are
used to make isoprenoids.
 The mevalonate pathway begins with acetyl-CoA and ends with the
production of IPP and DMAPP.
 These two building blocks add up to give different isoprenoids viz.,
monoterpenes, diterpenes, sesquiterpenes

13. Mevalonic Acid Pathway (MVA)
IPP & DMAPP ( Active isoprene units)
Isoprenoids
Terpenoids & Steroids
These precusor undergoes
condensation

14. 2 x Acetyl Co A Acetoacetyl CoA
Hydroxy methy CoA
( HMG CoA)
Mevalonate
5-
Phosphomevalon
ate
5- pyrophospho
mevalonate
Isopentyl pyrophosphate
(IPP) { C5 }
Dimethyl allyl pyrophosphate
(DMAPP) { C5 }
Geranyl pyrophosphate { C10 }
Farnesyl pyrophosphate { C15 }
(FPP)
Geranyl geranyl pyrophospate {
C20 }
Squalene { C30 }
1 2
3
45
7
8
9
10
AcetylCoA
thiolase
HMG CoA
synthase
HMG CoA
reductase
Mevalonate
kinase
ATP --> ADP
phosphorylatio
n
5-pyrophosphate
mevalonate
decarboxylase
6
11
IPP
Isomerase
Condensatio
n
+ IPP (C5)
+ IPP (C5) + FPP (C15)

16. Mevalonic Acid
DMAPP IPP
Geranyl pyrophospate (C10)
Farnesyl pyrophosphate
(FPP) (C15)
Squalene (C30) Geranyl geranyl pyrophosphate
(C20)
Monoterpenes
(menthol, citral)
Sesquiterpenes
(Santelene,
Zingiberene)
+FPP +IPP
Steroids
(Diosgenin)
Triterpenes
Diterpenes(
Taxol)
Polyterpene(rubber)
Tetraterpene (C40)
(carotenoids)
Gibberellins
phytol
+IPP
x2

17. ROLE OF MEVALONIC ACID
PATHWAY
 Biosynthesis of farnesyl pyrophosphate.
 Biosynthesis of Psoralens.
 Biosynthesis of rubber.
 Biosynthesis of diterpenoids.
 Biosynthesis of Gernylgeranyl pyrophosphate.
 Biosynthesis of cholesterol.
 Biosynthesis if Gibberellin.

18. Amino Acid Pathway
 Amino acids occur in plants both in the free state and as the basic
units of proteins and other metabolites.
 They are compounds containing one or more amino groups and
one or more carboxylic acid groups.
 Most of those found in nature are α-amino acids with an
asymmetric carbon atom and the general formula R–
CH(NH2)COOH.
 Some 20 different ones have been isolated from proteins, all having
an l-configuration.
R – CH – NH2
|
COOH

19.  Amino acids are also the precursors of some secondary metabolites.
 They arise at various levels of the glycolytic and TCA systems.
 Nitrogen appears to enter the metabolism of the organism by reductive
amination (1)of α-keto acids (pyruvic, oxalacetic and α-ketoglutaric acids)
 By transamination reactions (2) with other appropriate acids, alanine,
aspartic acid and glutamic acid serve as α-amino donors in the formation
of other amino acids.
a-keto acid + NH3 + NADPH Amino acid (x)+ NAD
Amino acid (y)
1
2

20. Reductive- and trans-amination in the
formation of amino acids.
HOOC-CH2 CH2-CO-COOH
(α -Ketoglutaric acid)
NH3
NH2
|
HOOC -CH2 -CH2 -CH-COOH
(Glutamic acid)
NH3
NH2
|
H2N-OC-CH2-CH2-CH-COOH
(Glutamine)
HOOC CO CH2 COOH
(Oxalacetic acid)
NH3
NH2
|
HOOC - CH2 -CH-COOH
(Aspartic acid)
NH3 NH2
|
H2N -OC -CH2 -CH-COOH
(Asparagine)
CH3 CO COOH
(Pyruvic acid)
NH3
NH2
|
CH3- CH-COOH
(Alanine)

22. Study of utilization of
radioactive isotopes in the
investigation of Biogenetic
studies.

23. Introduction
1. Living plants considered as biosynthetic laboratory primary as well
as secondary metabolite.
2. Different biosynthetic pathway:
» Shikmic acid pathway: Aromatic amino acids
» Mevalonic acid pathway: Terpenes
» Acetate pathway: Fatty acids
 Biosynthesis: Formation of a chemical compound by a living
organisms
 Biogenesis: Production or generation of living organisms from
other living organisms

24.  Various intermediate and steps are involved in biosynthetic
pathway in plants can be investigated by means of following
techniques: -
» Use of isolated organ
» Grafting methods
» Use of mutant strain
» Tracer technique
» Enzymatic studies

25. Isolated organ, tissue & cells:
 Growing of isolated organs , tissues & cells in suitable media or water is
one of the technique of investigation.
 This technique is useful in the determination of site of biosynthesis of
particular compounds.
 It eliminates interferences from other parts of plant which may produce
secondary change in metabolites.
 ex. Roots and leaves for the study of Nicotiana and Datura, petal disc for
the study of rose oil, tropane alkaloids in the root of solanaceae family.

26. Grafting methods:
 This technique uses joining two different but closely related plants together
to grow as one.
 The upper portion of on plant i.e., scion grafted in stock of another plant
 This method is used fore the study of alkaloid formation by grafted plants.
 ex. Tomato scions grafted on Datura produce alkaloids, while Datura scion
grafted on Tomato produce less quantity of alkaloids.
 This shows that main site of alkaloid biosynthesis is root.

27. Use of mutant strains:
 In this mutant strains of microorganisms are produced with the
lack of certain enzymes, which results in their metabolism
being blocked at a particular stage.
 ex. A mutant of Lactobacillus acidophilus, by its ability to
utilize a constituent of ‘brewer’s solubles’ but not acetate, led
to the isolation of mevalonic acid, an important intermediate of
the isoprenoid compound pathway.

28. Tracer technique:
 It can be defined as technique which utilizes a labelled compound to find
out or to trace the different intermediates and various steps in
biosynthetic pathways in plants, at a given rate & time.
OR
 In this technique different isotope, mainly the radioactive isotopes which
are incorporated into presumed precursor of plant metabolites and are used
as marker in biogenic experiments.

29. The labelled compound can be prepared by use of two types of isotopes.
» Radioactive isotopes:
• [e.g. 1H, 14C, 24Na, 42K, 35S, 35P, 131I decay with emission of radiation]
• For biological investigation – carbon & hydrogen.
• For metabolic studies – S, P, and alkali and alkaline earth metals are used.
• For studies on protein, alkaloids, and amino acid – labelled nitrogen atom
give more specific information.
• 3H compound is commercially available.
» Stable isotopes:
• [e.g. 2H, 13C, 15N, 18O]
• Used for labeling compounds as possible intermediates in biosynthetic
pathways.
• Usual method of detection are: – Mass spectroscopy [15N, 18O]
• NMR spectroscopy [2H, 13C]

30. SIGNIFICANCE OF TRACER TECHNIQUE
 Tracing of Biosynthetic Pathway: - e.g. By incorporation of
radioactive isotope of 14C into phenylalanine, the biosynthetic
cyanogenetic glycoside prunasin, can be detected.
 Location & Quantity of compound containing tracer: - 14C labelled
glucose is used for determination of glucose in biological system
 Different tracers for different studies: - For studies on nitrogen and
amino acid. (Labelled nitrogen give specific information than carbon)
 Convenient and suitable technique

31. CRITERIA FOR TRACER TECHIQUE
 The starting concentration of tracer must be sufficient withstand
resistance with dilution in course of metabolism.
 Proper Labelling: - for proper labelling physical & chemical nature of
compound must be known.
 Labelled compound should involve in the synthesis reaction.
 Labelled should not damage the system to which it is used.

32. Advantages
 High sensitivity.
 Applicable to all living
organism.
 Wide ranges of isotopes are
available.
 More reliable, easily
administration & isolation
procedure.
 Gives accurate result, if proper
metabolic time & technique
applied.
Disadvantages
 Kinetic effect
 Chemical effect
 Radiation effect
 Radiochemical purity
 High concentration distorting
the result.

33. REQUIREMENT FOR TRACER
TECHNIQUE
I. Preparation of labelled compound.
II. Introduction of labelled compound into a biological
system.
III. Separation & determination of labelled compound
in various biochemical fractions at later time.

34. I. Preparation of labelled compound.
o The labelled compound produce by growing chlorella in atmosphere of
14CO2 All carbon compounds 14C labelled.
o The 3H (tritium) labelled compound are commercially available. Tritium
labeling is effected by catalytic exchange in aqueous media by
hydrogenation of unsaturated compound with tritium gas. Tritium is pure β-
emitter of low intensity & its radiation energy is lower than 14C.
 By the use of organic synthesis: -
CH3MgBr +14CO2 CH3
14COOHMgBr + H2O
CH3
14COOH + Mg(OH)Br

35. II. Introduction of labelled compound
PRECAUTION: -
 The precursor should react at necessary site of synthesis in plant.
 Plant at the experiment time should synthesize the compound under investigation
 The dose given is for short period.
1) Root feeding: The plant in which roots are the biosynthetic sites e.g., Tobacco.
The plants are hydroponically cultivated to avoid microbial contamination.
2) Stem feeding: Substrate can be administered through cut ends of stem
immersed in solution. For latex containing plant this method is not suitable.

36. 3) Direct injection: Suitable for plant with hollow stem e.g., umbelliferous
and opium.
4) Infiltration: Suitable for plant rooted in soil or other support without
disturbing the roots (Wick feeding).
5) Floating method: When small amount of material is available this
method is used. This technique is used in conjugation with vacuum
infiltration to remove gases.
6) Spray technique: Compound has been absorbed after being sprayed
on leaves in aqueous solution e.g., Steroids

37. III. Separation and detection of
compound
 Geiger – Muller counter.
 Liquid Scintillation counter.
 Gas ionization chamber.
 Bernstein – Bellentine counter.
 Mass spectroscopy.
 NMR eletrodemeter.
 Autoradiography.
 Radio paper chromatography.

38. Geiger – Muller counter.
 A Geiger counter (Geiger-Muller tube) is a device used for the detection and
measurement of all types of radiation: alpha, beta and gamma radiation.
 Basically it consists of a pair of electrodes surrounded by a gas. The
electrodes have a high voltage across them. The gas used is usually Helium or
Argon. When radiation enters the tube it can ionize the gas. The ions (and
electrons) are attracted to the electrodes and an electric current is produced.
 A scaler counts the current pulses, and one obtains a "count" whenever
radiation ionizes the gas.

39. Liquid Scintillation counter.
 Scintillations literal meaning is luminescence and scintillators are material which
have property to luminate.
 When ionizing particles colloids on scintillating material they absorbs its energy
and exhibits scintillation ( i.e. re-emit the absorbed energy in the form of light)
 Light strikes on photosensitive surface, leads to release of photoelectron and
multiplication through the series of photo multiplier tubes(PMT).
 Light converts into an electrical signal.

40. Autoradiography
 Autoradiography is the bio-analytical technique used to visualize the distribution
radioactive labelled substance in a biological sample. It is a method by which a
radioactive material can be localized within a particular tissue, cell, cell organelles
or even biomolecules.
 In this method, the specimen under study is kept in contact with a suitable
photographic emulsion such as silver bromide gel or X-ray sensitive film for suitable
time period.
 After the film is developed in the usual manner and results are correlated with
darkening of the film.

41. METHODS IN TRACER TECHNIQUE
1. PRECURSOR PRODUCT SEQUENCE: - In this technique, the presumed
precursor of the constituent under investigation on a labelled form is fed into the
plant and after a suitable time the constituent is isolated, purified and radioactivity is
determined.
Disadvantage: -
The radioactivity of isolated compound alone is not usually sufficient evidence that the
particular compound fed is direct precursor, because substance may enter the general
metabolic pathway and from there may become randomly distributed through a whole
range of product.
Application: -
 Stopping of hordenine production in barley seedling after 15 – 20 days of
germination.
 Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura stramonium.
 This method is applied to the biogenesis of morphine & ergot alkaloids

42. 2. DOUBLE & MULTIPLE LABELLING
 This method give the evidence for nature of biochemical incorporation of
precursor arises double & triple labelling. In this method specifically labelled
precursor and their subsequent degradation of recover product are more
employed. Application: -
 This method is extensively applied to study the biogenesis of plant
secondary metabolite.
 Used for study of morphine alkaloid. E.g. Leete, use Doubly labeled lysine
used to determine which hydrogen of lysine molecule was involved in
formation of piperidine ring of anabasine in Nicotina glauca.

43. 3. COMPETITIVE FEEDING
 If incorporation is obtained it is necessary to consider whether this
infact, the normal route of synthesis in plant not the subsidiary
pathway. Competitive feeding can distinguish whether B & B’ is
normal intermediate in the formation of C from A.
Application: -
• This method is used for elucidation of biogenesis of propane alkaloids.
• Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis of
alkaloids of Conium maculactum (hemlock) using 14C labelled compounds.

44. 4. SEQUENTIAL ANALYSIS
 The principle of this method of investigation is to grow plant in
atmosphere of 14CO2 & then analyze the plant at given time interval
to obtain the sequence in which various correlated compound
become labelled.
Application: -
 14CO2 & sequential analysis has been very successfully used in
elucidation of carbon in photosynthesis.
 Determination of sequential formation of opium hemlock and tobacco
alkaloids.
 Exposure as less as 5 min. 14CO2, is used in detecting biosynthetic
sequence as –
 Piperitone --------- (-) Menthone ---------- (-) Menthol in Mentha
piperita.

45. APPLICATION OF TRACER TECHNIQUE
 Study of squalene cyclization by use of 14C, 3H labelled mevalonic acid.
 Interrelationship among 4 – methyl sterols & 4, 4 dimethyl sterols, by use of 14C
acetate.
 Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of 2- 14C
mevalonate.
 Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin.
 Origin of carbon & nitrogen atoms of purine ring system by use of 14C or 15N labelled
precursor.
 Study of formation of scopoletin by use of labelled phenylalanine.
 By use of 45Ca as tracer, - found that the uptake of calcium by plants from the soil.
(CaO & CaCO2).
 By adding ammonium phosphate labelled with 32P of known specific activity the
uptake of phosphorus is followed by measuring the radioactivity as label reaches
first in lower part of plant, than the upper part i.e. branches, leaves etc.