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3. Introduction to Secondary 3. Introduction to Secondary Metabolism and the Biosynthesis of Metabolism and the Biosynthesis of
Natural ProductsNatural Products
RA Macahig
FM Dayrit
PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES
CO2 + H2O Glucose
Polysaccharides
Pentose phosphateErythrose-4-phosphate
Phosphoenol pyruvate
Shikimate
Aromatic compounds(C6
-C1; C6-C2)
Phenylpropanoids (C 6-C3)
Lignans
PyruvateCitric acidcycle
Aromaticamino acids
Aliphaticamino acids
Aromatic alkaloids
Mixed alkaloids
Aliphatic alkaloids
Acetyl-CoA Polyketides Polyphenols
Phenylpropanoids
Flavonoids
Fatty acidsPolyacetylenesProstaglandins
Mevalonic acidTerpenesSteroidsCarotenoids
+NH3
Iridoids
Aliphaticamino acids
Alkaloids
3. Secondary metabolites and Biosynthesis (Dayrit) 2
Introduction Metabolism: (Gr. metabole = change) the totality of the chemical changes in living cells which involves the buildup and breakdown of chemical compounds.
Primary metabolism: biosynthesis, utilization and breakdown of the essential compounds and structural elements of the living organism, such as: sugars and polysaccharides; amino acids, peptides and proteins (including enzymes); fatty acids; and nucleotides. The starting materials are CO2, H2O and NH3. All organisms possess similar primary metabolic pathways and use similar primary metabolites.
3. Secondary metabolites and Biosynthesis (Dayrit) 3
Introduction Secondary metabolism: refers to the biosynthesis, utilization and breakdown of smaller organic compounds found in the cell. These compounds, called secondary metabolites, arise from a set of intermediate building blocks : acetyl coenzyme A (acetyl-CoA), mevalonic acid (MVA) and methyl erythritol phosphate (MEP), shikimic acid, and the amino acids phenylalanine/tyrosine, tryptophan, ornithine and lysine.
SCoA
O CO2H
CH3HO
OH
CO2H
OH
OH
HO
NH2R
CO2H
NNH2
CO2H
H
H2N CO2H
NH2
H2NCO2H
NH2
HO
CH3HO
OP
OH
3. Secondary metabolites and Biosynthesis (Dayrit) 4
Introduction Relationship between primary and secondary metabolism:
• The processes and products of primary metabolism are similar in most organisms, while those of secondary metabolism are more specific.
• In plants, primary metabolism is made up of photosynthesis, respiration, etc., using CO2, H2O, and NH3 as starting
materials, and forming products such as glucose, amino acids, nucleic acids. These are similar among different species. • In secondary metabolism, the biosynthetic steps, substrates and products are characteristic of families and species. Species which are taxonomically close display greater similarities (and metabolites); those which are distant have greater differences.
3. Secondary metabolites and Biosynthesis (Dayrit) 5
Introduction Biogenesis: overview of the origin of compounds starting from the set of intermediate building blocks: acetyl-CoA, MVA and MEP, shikimic acid, and the amino acids phenylalanine and tyrosine, tryptophan, ornithine and lysine.
SCoA
OCO2H
CH3HO
OH
CO2H
OH
OH
HO
NH2R
CO2H
NNH2
CO2H
H
H2N CO2H
NH2
H2NCO2H
NH2
Biosynthesis: detailed study of the step-wise formation of secondary metabolites. At more detailed levels, the specific enzymes, genes and signals are also identified.
HO
CH3HO
OP
OH
3. Secondary metabolites and Biosynthesis (Dayrit) 6
PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES
CO2 + H2O Glucose
Polysaccharides
Pentose phosphateErythrose-4-phosphate
Phosphoenol pyruvate
Shikimate
Aromatic compounds(C6
-C1; C6-C2)
Phenylpropanoids (C 6-C3)
Lignans
PyruvateCitric acidcycle
Aromaticamino acids
Aliphaticamino acids
Aromatic alkaloids
Mixed alkaloids
Aliphatic alkaloids
Acetyl-CoA Polyketides Polyphenols
Phenylpropanoids
Flavonoids
Fatty acidsPolyacetylenesProstaglandins
Mevalonic acidTerpenesSteroidsCarotenoids
+NH3
Iridoids
Aliphaticamino acids
Alkaloids
Overview of Secondary
Metabolism
* Metabolites found in
higher organisms only
*
*
*SCoA
O
CO2H
CH3HO
OH
CO2H
OH
OH
HO
NH2R
CO2H
NNH2
CO2H
H
H2N CO2H
NH2
7
Metabolite linkage map representing primary and secondary plant metabolism in opium poppy. The circles associated with each metabolite indicate whether the metabolite was detected (), not detected () or masked ().
(Zulak et al. BMC Plant Biology 2008 8:5; www.biomedcentral.com)
3. Secondary metabolites and Biosynthesis (Dayrit) 8
Biogenetic classification of natural products.
Biogenesis
Intermediate
Structural Types
Acetogenins (n x C2)
acetyl CoA
fats and lipids,
macrolides, phenols
Terpenoids (n x C5)
mevalonic acid, methyl erythritol phosphate
monoterpenes, sesquiterpenes, diterpenes, triterpenes, steroids
carotenoids
Shikimates
shikimic acid, prephenic acid
phenylpropanoids, phenols flavonoids
Aliphatic alkaloids
lysine, ornithine
aliphatic alkaloids
Aromatic alkaloids
phenylalanine, tyrosine,
tryptophan
aromatic alkaloids
3. Secondary metabolites and Biosynthesis (Dayrit) 9
The basic biogenetic and structural groups: Acetogenins
a. Acetogenins: Acetyl CoA fats, polyketides
CH3
CS
O
CoA = S-CoA
O
S-CoA
O
n x
CO2H
lauric acid
OHCH3
CO2H
6-methylsalicylic acid
3. Secondary metabolites and Biosynthesis (Dayrit) 10
The basic biogenetic and structural groups: Terpenoidsb. Isoprenoids: MVA terpenes, steroids; MEP carotenoids
=
CO2H
OH
H3C OH
"isoprene" mevalonic acid
n x
OH
menthol
HO
lanosterol
-carotene
HO
CH3HO
OP
OH
methyl erthritol phosphate
3. Secondary metabolites and Biosynthesis (Dayrit) 11
c. Shikimates: Shikimic acid phenylpropanoids
CO2H
OH
OH
HO
PO CO2-
OH
-O2C CO2
-
O
shikimic acid prephenatechorismic acid
CO2H
OH
O CO2H
p-hydroxybenzoic acid
CO2H
OH
CO2H
OH
OH
CO2H
NH2
R
caffeic acid R=H, phenylalanine
R=OH, tyrosine
The basic biogenetic and structural groups: Shikimates
3. Secondary metabolites and Biosynthesis (Dayrit) 12
d. Aliphatic alkaloids: Lysine aliphatic alkaloids
H2N CO2HH2N
ornithine
CH3N
OHtropine
e. Aromatic alkaloids: Phenylalanine aromatic alkaloids
phenylalanine
CO2H
NH2
ephedrine
N(H)CH3
HOCH3
NCH3
CH3
CH3O
HO
pellotine
The basic biogenetic and structural groups: Alkaloids
ExerciseExercise
The following cytotoxic anthraquinone derivative was recently isolated from the stem bark of Goniothalamus marcanii Craib. Propose its biogenetic origin. Highlight the appropriate atoms in the molecule.
N
O
O CH3
OCH3
O
OH
H
marcanin D
NCH3
CH3O
HO
CH3O
CH3O
OH
Propose its biogenetic origin of the following alkaloid. Highlight the appropriate atoms in the molecule.
Chemistry of Natural Products (Dayrit) 14
Exercises 2 & AnswersExercises 2 & Answers
The following cytotoxic anthraquinone derivative was recently isolated from the stem bark of Goniothalamus marcanii Craib. Propose its biogenetic origin. Highlight the appropriate atoms in the molecule.
Propose the biogenetic origin of the following alkaloid. Highlight the appropriate atoms in the molecule.
From Acyl-CoA From Methyl methionine
N
O
O CH3
OCH3
O
OH
H
marcanin D
From Methyl methionine
From Shikimate
7 AcylCoA’s + 2 methyl methionines
2 Phenylalanines/ Tyrosines + 2 methyl methionines
NCH3
CH3O
HO
CH3O
CH3O
OH
3. Secondary metabolites and Biosynthesis (Dayrit) 15
Phylogenetics and natural products
Prevalence of secondary metabolites in various organisms:• Bacteria and Fungi: Fats & lipids, Acetogenins, Terpenes• Plants: +Phenylpropanoids, +Alkaloids
Variations of secondary metabolism exist in various organisms. For example, recently a second pathway in the biosynthesis of terpenes in plants was discovered. The first pathway is the better-known mevalonic acid (MVA) pathway; the second pathway is the methyl erythritol phosphate (MEP) pathway which operates in the chloroplast.
Many of the early biosynthetic studies were conducted using bacteria, in particular E. coli. It is possible that processes in higher organisms differ, and that revisions may appear in the future.
3. Secondary metabolites and Biosynthesis (Dayrit) 16
Phylogenetics and natural products:
Evolution of terpene biosynthesis in plantsAcetate
Mevalonate
C10 Iridoids Indole alkaloids(Labiatae) (Apocynaceae)
C15 Sesquiterpenes Sesquiterpene lactones(Myrtaceae) (Compositae)
C20 Diterpenes Diterpene acids(Euphorbiaceae) (Leguminosae)
C30 Steroidal alkaloids(Solanaceae)
3. Secondary metabolites and Biosynthesis (Dayrit) 17
Evolution of secondary metabolism in higher plants (http://www.uk.plbio.kvl.dk/plbio/students-projects/evolution-sec-metaboites.pdf)
• Cytochromes P450 and family 1 glycosyltransferases are key enzymes in biosynthesis of secondary metabolites found in higher plants. Genomic and cDNA sequencing programs of a number of model plants have unravelled a wealth of information on genes and genomes giving better understanding of evolution in terrestrial plants.
• Deduced sequences of genes can be used in the analysis of phylogenetic trees to obtain their evolutionary relationship.
3. Secondary metabolites and Biosynthesis (Dayrit) 18
This section will focus on the chemical transformations of biosynthesis. It will also survey the enzymes which are responsible for these transformations.
Introduction to Biosynthesis
Natural products are unparalleled in the diversity and complexity of chemical structures. Despite the complexity of natural products, it should be emphasized that biosynthesis proceeds by discrete chemically reasonable steps. That is, no matter how complicated a natural product compound is, one can rationalize its biosynthesis using a series of simple chemical transformations,.
3. Secondary metabolites and Biosynthesis (Dayrit) 19
Why study the biosynthetic pathway?
• The determination of the biosynthetic pathway enables us to understand the relationships and dynamic flow of the compounds that are present in a living cell.
• The objective of the study of a biochemical sequence is to be able to identify the “intermediates” and the “product”. However, there are cases when this is not so obvious. During the chemical extraction process, we obtain many of these compounds and the problem is to determine the sequence of their formation.
• An understanding of a biosynthetic sequence can help us identify the enzymes and genes, understand the relationships among different organisms (such as symbiosis, plant-insect interactions, etc). An understanding of biosynthesis is part of a complete understanding of plant biology, ecology and biodiversity.
3. Secondary metabolites and Biosynthesis (Dayrit) 20
An understanding of biosynthesis is very useful!
• It enables us to classify the diversity and complexity of natural products structures.
• It reveals the functional relationships among natural products in a dynamic context.
• It provides essential information which enables us to control or manipulate the formation of desired metabolites.
• It opens up possible directions in biotechnology and molecular biology through the study of enzymes (proteomics) and genomics:
Genomics + Proteomics + Biosynthesis = Metabolonomics
21
Some types of biosynthetic pathways: 1. Simple linear process A B C ..... X Y
2. Modified linear process
A B Y Z
C D
M N
3. Convergent process A B C
D E
Y
4. Branching process A B C D .......... Y
E
F
G
5. Metabolic grid A B C
D E F
G H Y3. Secondary metabolites and Biosynthesis (Dayrit)
3. Secondary metabolites and Biosynthesis (Dayrit) 22
Some comments on biosynthetic pathways:
1. A compound is an obligatory intermediate if its formation is required for the biosynthetic process to continue and there are no alternative pathways. Such is the case for the compounds in a linear pathway. In comparison, a metabolic grid provides many alternative routes to the product.
2. Although compounds are usually transformed from simple structures to more complex ones, this is not always the case.
YX.....CBA
C
BA
D
Y Z
NM
CBA
D FE
H YG
3. Secondary metabolites and Biosynthesis (Dayrit) 23
Some comments on biosynthetic pathways:
3. Different organisms may produce the same types of compounds through different pathways (e.g., convergent evolution), even if they are widely separated phylogenetically.
4. Some compounds may be produced by the same organism via more than one biosynthetic path. That is, there may be more than one path available, such as in a modified linear process or metabolic grid.
5. Even if the same compound is present in two different organisms, it is possible that they are formed via different pathways. This, however, is more likely for metabolites with simple structures.
3. Secondary metabolites and Biosynthesis (Dayrit) 24
Some comments on biosynthetic pathways:
6. The production of secondary metabolites depends on genetic and environmental factors. That is, secondary metabolites may be present in the organism in various amounts depending on the time of day or season, at particular stages of the organism’s life, or in response to certain environmental stimuli (e.g., production of defense compounds).
7. Because these compounds are produced by specific enzymes and precursors, it can be assumed that they are produced in specific parts or organelles of the plant.
8. Secondary metabolites are probably in a state of dynamic flux, being produced and broken down constantly. Some compounds, however, may be stored in specific organelles and have more constant presence.
3. Secondary metabolites and Biosynthesis (Dayrit) 25
General strategies for studying secondary metabolism:
1. Enzyme control. If the enzymes in the biosynthetic pathway are known or have been isolated, these enzymes can be blocked either by introducing enzyme inhibitors or by causing mutations which alter the activities of these enzymes.
2. Metabolite control. Many secondary metabolites are controlled by a feedback mechanism. It is reasonable to assume that there is a steady-state condition operating in the organism where the concentrations of the metabolites are maintained at some level. Effect on biosynthesis may be negative (inhibitory) or positive.
3. Secondary metabolites and Biosynthesis (Dayrit) 26
Strategies for studying secondary metabolism: Enzyme control
Experiment Biosynthetic process Comments
Overall process A B C DEa Eb Ec
Exp. 1 EaA B C Dx x x A accumulates when enzyme Ea is
blocked; B, C and D are not formed
Exp. 2 Ea Eb A B C Dx x B accumulates when enzyme Eb is
blocked; C and D are not formed
Exp. 3 Ea Eb EcA B C Dx C accumulates when enzyme Ec is
blocked; D is not formed
Example: the biosynthetic sequence in a linear process using mutants or enzyme inhibitors
3. Secondary metabolites and Biosynthesis (Dayrit) 27
Type Isotope used Method ofDetection
Comments
Radioactive 3H, 14C scintillation Advantages: High sensitivity, requires only asmall amount of material
Disadvantage: special procedures requireddue to radioactivity
Non-radioactive
2H, 13C, 19F NMR, MS Advantage: Structural information available
Disadvantages: Relatively lower sensitivity;expensive instrumentation
Strategies for studying secondary metabolism: Metabolite control
3. Secondary metabolites and Biosynthesis (Dayrit) 28
Examples of isotopically-label compounds used in biosynthetic studies:
..= 13C or 14C
H3CS CO2H
NH2
.methionine
H3CC
OH
O
D3CC
OH
O
H3CC
OH
O..acetic acid
-O2C OP
CH3HO
.mevalonate
2 5
CO2H
NH2
.
phenylalanine
52
.-O2C OP
CH3HO
DD
-O2C OP
CH3HO
DD
2 5
3. Secondary metabolites and Biosynthesis (Dayrit) 29
Examples of isotopically-label compounds used in biosynthetic studies:
a. Skimmianine, in Choisya ternata (Grundon, Harrison and Spyropoulos, Chem. Comm., 51, 1974).
N
H
O
TTCH3O
N
CH3O
O
T. .
3H : 14C = 2 : 1
Skimmianine
3H : 14C = 1.1 : 1
3. Secondary metabolites and Biosynthesis (Dayrit) 30
Examples of isotopically-label compounds used in biosynthetic studies:
b. Ephedrine, in Ephedra distachya (Yamasaki, Sankawa and Shibata, Tetrahed. Lett., 4099, 1969).
CO2-
NH3+
T5
.T5
OH
CH3
N(H)CH3
D,L-phenylalanine (-) ephedrine
[14C = nil]
c. Tyrosine, in Psuedomonas (Bowman, Gretton and Kirby, J. Chem. Soc. Perkin I, 218, 1973).
CO2-
NH3+
T
. CO2-
NH3+
HO
T phenylalanine
tyrosine
.
3. Secondary metabolites and Biosynthesis (Dayrit) 31
Major chemical transformations in Biosynthesis
1. Hydrolysis2. Esterification
3. Oxidation
4. Reduction
5. C-C Bond formation
6. Nucleophilic substitution
7. Elimination reaction
8. Cationic rearrangement
3. Secondary metabolites and Biosynthesis (Dayrit) 32
Major biosynthetic transformations
Reaction Classification
General equation Comments
1. Hydrolysis
R1 OR2
O
R1 OH
O+ R OH2
Common transformation.
2. Esterification
R1 OH
O+ R OH2
R1 OR2
O
Common transformation.
3. Oxidation
a. C-H C-OH [ OH]
R1 R2
HaHb.
R1 R2
OHHb
Generally stereospecific.
b. Epoxidation [O]
OGenerally stereospecific
ReactionClassification
General equation Comments
c. Double bondoxidation
R1 R3
R4R2
[2 O] R1
R2
O
R4
R3
O
3. Secondary metabolites and Biosynthesis (Dayrit) 33
Major biosynthetic transformationsReaction
ClassificationGeneral equation Comments
d. Dehydrogenation H
H
H
H
-2HH
H
e. Halogenation H Cl
4. Reduction
a. e- transfer + H+ H
H
+2H
H
H
H
H
[H] = e- transfer, then + H+
b. deoxygenation
R1 R2
O
R1 R2
OHH
R1 R2
HH
3. Secondary metabolites and Biosynthesis (Dayrit) 34
Major biosynthetic transformations
ReactionClassification
General equation Comments
5. C-C bond formation
a. Radical coupling Commonly observedin aromatic andconjugated systems
OH
-H.
O . O
.
couplingHO OH
b. Claisencondensation
R2
O
R1 R2
O
R1
R3O
R COX
base3
+ X_
Very common reaction,e.g., in lenghtening ofpolyketide chain
c. Aldol
R1R2
O
+ R3 H
O
base
R1R2
O
R3 OH
base
R1R2
O
R3
3. Secondary metabolites and Biosynthesis (Dayrit) 35
Major biosynthetic transformations
ReactionClassification
General equation Comments
6. Nucleophilic substitution, Sn2
CH3
SCH2
-CH2-CH(NH2)CO2H
R
+
+H
OR1 CH3
OR1
Conversion of alcohol to methyl ether. Methyl methionine is usual methyl source.
7. Elimination reaction, E2 R2
R1
OH
H
baseR2
R1
-OH is usually converted to –OPP which becomes leaving group
8. Cationic
rearrangement
a. 1,2-methyl migration
CH3
H
CH3+
+
b. Wagner-
Meerwein shift
+ +
Common in monoterpenesand sesquiterpenes.
3. Secondary metabolites and Biosynthesis (Dayrit) 36
Major biosynthetic transformations
ReactionClassification
General equation Comments
9. Orbital symmetry-controlled
a. 3,3,-sigmatropic shift
O1
23
32
1 12
3
32
1O
Not commonly observed.
10. Carboxylation
R1R2
O
baseCO 2 R1
R2
O
CO2-
Commonly observed in activation of -position for nucleophilic attack.
11. Decarboxylation R1
R2
O
CO2-
2-COR1
R2
O
Usually observed together with carboxylation to remove carboxylic activating group.
3. Secondary metabolites and Biosynthesis (Dayrit) 37
Most of the biosynthetic reactions are mediated by specific enzymes. Enzymes have five fundamental properties:
Enzymes in biosynthesis
1. increase in reaction rate - enzymes are catalysts which increase the forward and reverse rates of a chemical step.
2. kinetic control - Enzymes are subject to various types of control, such as pH and feedback.
3. chemoselectivity - Enzymes can distinguish functional groups. For example, in an oxidation reaction: C-H C-OH, chemoselectivity allows the differentiation between various types of C-H, such as primary, secondary and tertiary alkyl, olefinic and aromatic positions.
3. Secondary metabolites and Biosynthesis (Dayrit) 38
Enzymes in biosynthesis
4. regioselectivity - Regioselectivity is the ability of select only one site of reaction from a number of possibilities of the same functional group. For example, in a long chain saturated fatty acid, the initial site of dehydrogenation is typically 9,10. In a sugar, or a compound with many -OH groups, the position of methylation is specific.
5. stereoselectivity - This refers to the chiral recognition of substrates (compare with chemoselectivity).
3. Secondary metabolites and Biosynthesis (Dayrit) 39
Stereoselectivity in biosynthesis
Classification of stereoselectivity:
• Enantioselective - The reactants are enantiomeric and the enzyme reacts with only one enantiomer.
• Prochiral - The carbon reaction center, CH2(R1)(R2), is not chiral, but becomes chiral with substitution of one of the hydrogens. In the case of a ketone, (R1)(R2)C=O, where R1R2, reduction of the carbonyl to an alcohol produces a chiral center at the carbon.
R1 R2
HaHbpro-Spro-R
OR1
R2
re-face
si-face
3. Secondary metabolites and Biosynthesis (Dayrit) 40
Control of enzyme activity
• An organism must be able to regulate its enzymes so that it can coordinate its many biosynthetic activities and respond to its environment. It is reasonable to assume that the organism derives an advantage or fulfills a need when it biosynthesizes secondary metabolites. Therefore, careful control of their biosynthesis is an important ability.
• There are two major types of control of biosynthesis: • inhibition of a specific enzyme by one of the
metabolites (protein inhibition); and • regulation by induction or repression of gene
expression.
3. Secondary metabolites and Biosynthesis (Dayrit) 41
Inhibition of enzyme activity
• Feedback inhibition is one common mode of biosynthetic regulation in which the changing concentration of a product attenuates (decreases) the activity of an enzyme.
• Allosteric control (Greek: allos, other + stereos, space or solid) occurs when the binding of the substrate is selectively increased or decreased by the binding of another species at a different (allosteric) site on the enzyme.
3. Secondary metabolites and Biosynthesis (Dayrit) 42
Types of feedback control of biosynthesis.
1. Simple mass action: In a reversible process, if the ratio of the concentrations of products over those of reactants, [P]/[R], is not equal to the equilibrium constant, K, then the equilibrium will shift accordingly.
2. Reversible competitive inhibition of the enzyme by the product: In this case, the product slows down its own formation by inhibition of the enzyme.
3. Product or reactant interacts with the DNA or RNA to induce or repress the synthesis of the enzymes which are responsible for the biosynthesis.
3. Secondary metabolites and Biosynthesis (Dayrit) 43
Some types of control of
biosynthetic activity through
the action of metabolites on
enzymes.
A. Negative feedback by one of the products: A B C D
B. Negative feedback by a combination D
of products: A B C }
E
C. Selective positive / negative feedback by products:
C D
A B
E F
(-)
D+E
(-)
D
D
(-)
(+)
F
D. Allosteric control: E(+)=enzyme in active form;
E(-)=enzyme in inactive form; A=substrate; B= product;
P=positive effector; N=negative effector
E(-)
E(+)
N P
A B
3. Secondary metabolites and Biosynthesis (Dayrit) 44
Schematic representation of the mechanisms for inducing or repressing gene
function.
Chromosome
Operator Gene 1 Gene 2 Gene 3
Enzyme 1 Enzyme 2 Enzyme 3
A B C D
A. General mechanism
B. Control by induction of transcription of enzyme synthesis by I.
OperatorOperator + I OperatorOperator - I
(inactive biosynthesis) (active biosynthesis)
(inactive enzyme
degradation)
(active enzyme
degradation)
Operator - IOperator - R+ ROperatorOperator
C. Control by repression of enzyme degradation by R.
3. Secondary metabolites and Biosynthesis (Dayrit) 45
Enzyme classification (EC) system
Classification (EC) Type of reaction catalyzed
1: Oxidoreductase oxidation-reduction: transfer of e- from a donor which is
oxidized to an acceptor which is reduced
2: Transferase transfer of functional groups
3: Hydrolase hydrolysis, for example, of ester or amide groups, oresterification
4: Lyase elimination of a group of adjacent groups of atoms to form adouble bond, or addition of a group of atoms to a doublebond
5: Isomerase conversion of a compound into its isomer
6: Ligase bond formation accompanied by ATP hydrolysis; also knownas synthetase
3. Secondary metabolites and Biosynthesis (Dayrit) 46
The IUB number and classification of enzymes Main Classes and Subclasses Main Classes and Subclasses
1: Oxidoreductase 1.1: acts on the CH-OH group of donors 1.2: acts on the aldehyde or keto group of donors 1.3: acts on the CH-CH group of donors 1.4: acts on the CH-NH2 group of donors 1.5: acts on the C-NH group of donors 1.6: acts on (reduced) NADH or NADPH as a donor
of H-
1.7: acts on other nitrogenous compounds as donor 1.8: acts on sulphur groups as donor 1.9: acts on haem groups as donor 1.10: acts on diphenols and related substances as
donor 1.11: acts on H2O2 as electron acceptor 1.12: acts on H2 as donor 1.13: acts on single donors with incorporation of
oxygen (oxygenases) 1.14: acts on paired donors with incorporation of
oxygen into one donor (hydrolase).
2: Transferase 2.1: transfers one-carbon group 2.2: transfers aldehyde or ketone 2.3: acyltranferase 2.4: glycosyltransferase 2.5: transfers other alkyl groups 2.6: transfers nitrogenous groups 2.7: transfers phosphorous-containing groups 2.8: transfers sulphur-containing groups
3: Hydrolase 3.1: hydrolysis of the ester bond 3.2: hydrolysis of the glycosyl bond 3.3: hydrolysis of the ether bond 3.4: hydrolysis of the peptide bond 3.5: hydrolysis of C-N bond other than the peptide
bond 3.6: hydrolysis of the acid-anhydride bond 3.7: hydrolysis of C-C bond 3.8: hydrolysis of the C-halide bond 3.9: hydrolysis of the P-N bond
4: Lyase 4.1: lysis of C-C bond 4.2: lysis of C-O bond 4.3: lysis of C-N bond 4.4: lysis of C-S bond 4.5: lysis of C-halide bond 4.99: others
5: Isomerase 5.1: racemization and epimerization 5.2: cis-trans isomerization 5.3: intramolecular oxidoreduction, e.g. aldehyde-
ketone, keto-enol, double bond migration 5.4: intramolecular group transfers 5.99: other isomerizations
6: Ligase 6.1: formation of C-O bond 6.2: formation of C-S bond 6.3: formation of C-N bond 6.4: formation of C-C bond
3. Secondary metabolites and Biosynthesis (Dayrit) 47
The four major types of biological oxidation reactions catalyzed by oxidoreductases
Type ofOxidation
Description Schematic Reaction and Examples
D ehydrogenase R em oves o f tw o H a tom s from thesubstrate, and transfers th is toanother organic com pound. The H -acceptor, A , is a coenzym e.
SH 2 + A S + A H2
RC H 2
C H2R
R R
H H
R
C H O H
R
R
C O
R
R R
H HO
RC H 2
C H 2R
Oxidase Removes two H atoms from thesubstrate and utilizes O2 or H2O2 asthe H-acceptor.
SH2 + ½O2 S + H2O
SH2 + H2O2 S + 2H2O
OH
OH
O
O
2O1/2
3. Secondary metabolites and Biosynthesis (Dayrit) 48
The four major types of biological oxidation reactions catalyzed by oxidoreductases
Type ofOxidation
Description Schematic Reaction and Examples
M onooxygenase A dds one O a tom to the substra te. Ais a coenzym e.
S + A H 2 + O 2 S O + A + H 2OR R
H H
R R
H HO
C H RC H2
O H
RR
C H2
C H2R
RC
H
O
RC
O H
O
Dioxygenase Adds two O atoms to the substrate S + O2 SO2
R1 R2
H H
O2R1
H
O
R2
H
O+
3. Secondary metabolites and Biosynthesis (Dayrit) 49
Elimination and rearrangement reactions following oxidation
RO
CH3 RO
CH2O-H R OH + HCHO
[O]
A. Demethylation: Methyl ether to alcohol
[O] + HCHOR1
NR2
CH3CH2
O-HNR1
R2
R1NH
R2
B. Demethylation: Methyl amine to amine
C. Formation of phenyl methylenedioxy ring
O-CH3
OH
[O]O-CH2
OH
OH O
CH2
O
-H2O
3. Secondary metabolites and Biosynthesis (Dayrit) 50
Elimination and rearrangement reactions following oxidation
D. Aromatic ring opening reaction (mono-oxygenase)
[O]O O
E. Aromatic ring opening reaction (dioxygenase)
[O ]OH
OH
2OH
OH
OO
+
_ OH
OH
OO
H
CO2HCHO
OH
F. Oxidation of aromatic ring: NIH shift (hydride shift); R = alkyl group
O
R
D
H
[O]
R
D
R
O
H
DR
OH
D
isotopeeffect
hydride shift
3. Secondary metabolites and Biosynthesis (Dayrit) 51
Elimination and rearrangement reactions following oxidation
R-O
OH
R-O
H
O
R-O
[O]O
R-O H
H
G. Para oxidation of aromatic ring.
_+
H. Oxidative decarboxylation of aromatic carboxylic acid.
[O]CO2
_ _
O
OO
-CO2OH
3. Secondary metabolites and Biosynthesis (Dayrit) 52
Oxidative coupling of phenols
OH
H3C
A. Illustration of phenoxy radical formation, resonance stabilization and coupling: Pummerer's ketone.
baseO
H3C
_
-e_ O
H3C
H
.
.
O
H3C H
O
H3C.
.O
H3C
.
O
H3C
O
H3C H. +
O
H3C
O
H CH3
OH
H3C
O
CH3
O
H3C
OH
CH3
3. Secondary metabolites and Biosynthesis (Dayrit) 53
Oxidative coupling of phenols B. Some important phenolic structures which can undergo phenolic coupling.
OH OH
*
**
* *
*
*
*
HO O
O
OH
OHHO*
*
OH
CH3
CHO
HO
HO
H3C CH3
CO2H
OH
HO
HO CH2OH
HO
O-CH3
*
**
3. Secondary metabolites and Biosynthesis (Dayrit) 54
Carbon-carbon bond formation by Sn2 displacement of a stable nucleophile on an electrophilic alkylating agent. A. Methylation of alcohol or amine with S-adenosyl-L-methionine as alkylating agent..
R OH H3C
S
(Adenosyl)
H2N
CO2H
+ -H+
R OCH3
B. Glycosylation of an alcohol with glycosyl phosphate as alkylating agent.
OOH
OHHO
HOOP
HO R
GlyO
R
3. Secondary metabolites and Biosynthesis (Dayrit) 55
Carbon-carbon bond formation by Sn2 displacement of a stable nucleophile on an electrophilic alkylating agent. C. Alkylation of a stabilized carbanion with acetyl CoA as alkylating agent.
R CH2
O
O
O
_-CO2
_
R CH2
O
R CH2
O_
H3C S-CoA
O R CH3
OO
OPP
D. Sn2 displacement of pyrophosphate.
OPP
H H-H , -OPP
+ _
OPP
Note: One common series of reactions for Sn2 displacement is:• phosphorylation of R-OH group R-OPP-, followed by• Sn2 displacement of OPP- by nucleophile.
3. Secondary metabolites and Biosynthesis (Dayrit) 56
Control of biosynthesis in plants
Plants exercise control over the biosynthesis in several ways:
• First, the enzymes are coded for separately allowing better control of each enzyme.
• Second, several of the enzymes exist in more than one form. It is believed that the existence of isozymes allows the plant better regulation of biosynthesis.
• Third, some of the biosynthetic transformations can take more than one pathway.
3. Secondary metabolites and Biosynthesis (Dayrit) 57
Control of biosynthesis in plants: alternative pathways to tyrosine (a modified linear process)
OH
CH2CCO2H
O
OH
NH2
CH2CHCO2H
HO2C CH2CCO2H
OH
O
HO2C CH2CHCO2H
OH
NH2
Prephenic acid
4-Hydroxyphenylpyruvic acid
Tyrosine
Pretyrosine
prehenatedehydrogenase,NAD+
4-hydroxyphenylpyrivatetransaminase, pyridoxal-5'-phosphate
4-hydroxyphenylpyrivatetransaminase, pyridoxal-5'-phosphate
pretyrosinedehydrogenase,NAD+
3. Secondary metabolites and Biosynthesis (Dayrit) 58
Localization of enzymes
• One of the important phenomena of living organisms is cell structure and differentiation. This means that many functions of cells are localized in certain parts of the cell and that different types of cells within the same organism have different functions.
• Enzymes of different types can be found in all parts of the cell. While many types of enzymes are assumed to function in the cytosol, some enzymes are known to be localized in specific parts of the cell and be active only under certain conditions.
3. Secondary metabolites and Biosynthesis (Dayrit) 59
Localization of enzymes
• One well studied system is fatty acid synthase. Fatty acids play different roles in the organism. First, fatty acids are a form of energy storage; second, fatty acids are essential constituents of the cell membrane; third, fatty acids are sometimes found to be components of other natural products (R-OH) being attached as esters.
• Consistent with this observation, the synthesis of fatty acids takes place in three different sites of the cell and is mediated by three enzymatic systems: the mitochondrial system, the cytoplasmic system, and the microsomal system.
• We will discuss this further when we cover fats.
3. Secondary metabolites and Biosynthesis (Dayrit) 60
Comments regarding biosynthetic mechanisms
There are three approaches to the study of natural products:
• Classification of natural products according to activity, such as pharmacological activity (e.g., antioxidants) or ecological function.
• Classification based on structural types and physico-chemical properties, for example, phenolics, glycosides, etc.
• Classification according to biogenetic origins or biosynthetic pathways.
3. Secondary metabolites and Biosynthesis (Dayrit) 61
Advantages of the approach of biosynthesis
• It follows established principles and mechanisms of organic chemistry.
• This approach readily links with the fields of biochemistry, genetics, ecological interactions and evolutionary development.
• It also provides insight into the structural relationships among secondary metabolites.
The biosynthetic mechanism can be used to guide further research into the search for enzymes and genes.
3. Secondary metabolites and Biosynthesis (Dayrit) 62
Tips on biosynthetic mechanisms
How does one judge a “good” from a “bad” biosynthetic mechanism?
1. A good mechanism is based on precedent: it should follow patterns of known biosynthetic transformations.
2. If appropriate, the mechanism should start with intermediate metabolites which are already well known.
3. It should use known enzymatic transformations.
4. There should be economy of reaction.
5. The transformations should not be too cluttered.
3. Secondary metabolites and Biosynthesis (Dayrit) 63
Summary
1. All secondary metabolites, no matter how complex, are biosynthesized via discrete chemically-reasonable steps. The biosynthetic transformations are classified as follows: 1. hydrolysis 2. esterification3. oxidation: hydroxylation, epoxidation or oxygenation of alkene,
dehydrogenation, halogenation 4. reduction: hydrogenation, deoxygenation5. carbon-carbon bond formation: aromatic radical coupling,
Claisen condensation, aldol condensation 6. Cationic rearrangement: 1,2-migration, Wagner-Meerwein7. Rearrangement under control of orbital symmetry 8. Sn2 displacement 9. E2 elimination 10. carboxylation / decarboxylation
3. Secondary metabolites and Biosynthesis (Dayrit) 64
Summary
2. Each step is presumed to be mediated by a specific enzyme. All chemical transformations are accounted for by the system of six enzyme classes: 1. oxidoreductase2. transferase 3. hydrolase 4. lyase 5. isomerase6. ligase
3. The enzymes are located in specific parts of the cell, and in some cases may be immobilized on a membrane.
4, The enzymes are coded for in the plant’s genome whose expression can be controlled at the level of the gene.