Alkaloid Biosynthesis:

Me
Nitrogen containing compounds

CO2Me

NH3
O

cocaine
Starting materials:
Amino acids and nucleic acids

N
H Me

OH
H

CO2
NH3

NMe
H

H3N
CO2

N
H

HN

ornithine

ergot

tryptophan

nicotine
NH2

O
H3C

CH3
N

N
O
-O P O
O-

N
N
CH3

purine

Me

pseudopelletierine

NCH3

HO

morphine

tyrosine

CO2

NH3
CO2

lysine

OH

N H
Me

H3N
CO2

HO

OH

HO

NH3

O
OH OH

caffeine

ajmaline

NH3
N
H

tryptophan
S. OConnor
Biochemistry 704
2/5/08

Endogenous Roles of Natural Products

CHCl2
HO

OH

H3C

OH

CH3
N

N
N
CH3

UV-protectants

N
H Me

O
HO

NH
OH

NO2

insecticide

antibiotic/antifungal
OH

OCH3
OH
O
HO

O
HO
HO

color

HO
O
OH

N
N

O
OH

O
O

O
HO

H3CO
Cl

OH

signalling
HO
OH

Cl
OH

HO

scent

attraction

S. OConnor
Biochemistry 704
2/5/08

Endogenous Roles of Natural Products

alkaloids made by
plants
Me

CO2Me

O
O

cocaine

fungi
O

OH
H
NMe
H

HN

ergot

bacteria
S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinolin
HO
e Biosynthesis

HO
NH2

HO

L-tyrosine

NCS

HO

N+

HO

BBE

OMe

H3CO

OCH3

(S)-scoulerine

berberine type
O

H3CO

NCH3+

H3CO
NCH3
H

HO
HO

H
H3CO

H3CO

HO

SAT

NCH3

thebaine

codeinone
CO R

HO

NCH3

NCH3

H3CO
HO

salitidarol-7-O -acetate

salutaridinol

HO

NCH3 H3CO

H
H3CO
HO

H3CO
O

salutaridine
SO R

NCH3

oxycodone

NCH3

(R)-reticuline

O
H

HO

H3CO

H3CO

spontaneous
HO

STS

H3CO

H3CO

HO

H3CO

1,2-dehydroreticuline

benzophenanthridine type
O
sanguinarine

NCH3
H

HO
HO

(S)-reticuline

H3CO

4O M T

NCH3
H

HO
HO

N+

HO

CPY80B3

H3CO
O

NCH3
H

HO

H3CO

HO
HO

OH

OMe

berberine

NH
H

HO

H3CO
N

N MT

HO

(S)-norcoclaurine
H3CO

6-OMT

NH
H

HO

O
HO

H3CO

H3CO

O
H

NCH3

HO

HO

codeine

morphine

NCH3

morphanan type

S. OConnor
Biochemistry 704
2/5/08

How are new genes discovered??

1.

Genome Mining (for sequenced prokaryotes/fungi)

2.

Reverse genetics

3.

Homology cloning (P450s, acetyltransferases, terpene synthases)

4.

Large scale sequencing

seqeunce all (Taxol)
sequence cell type (tabersonine OMe transferase- next page)

5.

Functional expression

6.

RNAi screens

S. OConnor
Biochemistry 704
2/5/08

How are new genes discovered??

expressed in leaf epidermis

N H

N
H

N H

T16H
HO

CO2Me

N
H

HTOM
MeO

N
H

CO2Me

16-hydroxy-tabersonine

tabersonine

N H

N H
MeO

CO2Me

16-methoxy-tabersonine

CO2Me
N
H H OH

16-methoxy-2,3dihydro-3hydroxytabersonine
NMT

N H
MeO

N
Me H OH

vindoline

OAc
CO2Me

N H

DAT
MeO

N
Me H OH

OH
CO2Me

deacteylvindoline

N H

D4H
MeO

CO2Me
N
Me H OH

desacetoxyvindoline

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis

CO2HO

HO
NH2

HO

NH3+

dopamine

L-tyrosine

HO

NCS

HO

MeO
NH
H

6-OMT

HO

NH
H

O
H

HO

p-hydroxyphenylacetaldehyde

MeO
HO
HO

HO

HO

(S)-norcoclaurine
NMT

MeO
NMe
H

MeO

4'-OMT

HO
HO
HO

MeO
NMe
H

NMTC

HO

NMe
H

HO

(S)-reticuline

J Nat Prod (2005), 68, 666-673.

S. OConnor
Biochemistry 704
2/5/08

How determine the order of the steps in a pathway?

track biosynthetic intermediates before and after knocking out genes

assay enzymes in vitro with different substrates

J Nat Prod (2005), 68, 666-673.

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis
Two oxidative paths

MeO

MeO
NMe
H

HO
HO

BBE

MeO
N

HO

SOMT

berberine

OH

MeO

OMe

(S)-tetrahydrocolumbamine

OMe

(S)-reticuline

MeO

(S)-scoulerine

OMe

RO
MeO
HO
HO

MeO

MeO
NMe

MeO

1,2-dehydroreticulinium ion

DHR

HO
HO

NMe
H

MeO

(R)-reticuline

STS

HO

morphine
H

NMe

MeO
O

salutaridine

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis

MeO
HO
HO

MeO
NMe
H

BBE

HO

MeO

MeO
N

SOMT

OH

(S)-scoulerine

(S)-reticuline

MeO

OMe

N
H

OMe

(S)-tetrahydrocolumbamine

OMe

CS
O

O
O

N+

TBO
OMe

berberine

OMe

N
H

(S)-canadine

OMe
OMe

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis

JBC (1995) 270 24475-24481

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis
MeO

MeO
NMe
H

HO
HO

RO

MeO

DHR

NMe

HO
HO
MeO

(S)-reticuline

NMe
H

HO
HO

spontaneous

NMe

MeO

SAT

HO

NMe

H
MeO
HO

MeO

neopinone

thebaine

MeO

O
H

codeinone

H
MeO
HO

NMe

salutaridinol

HO

demethylation
O

NMe

HO

NMe

salitidarol-7-O-acetate

MeO

COR

salutaridine
SR

O
H

NMe

MeO

MeO

demethylate

HO
H

(R)-reticuline

MeO

STS

MeO

1,2-dehydroreticulinium ion

MeO

MeO

MeO

O
H

HO

NMe

NMe

HO

codeine

morphine
S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis
STS
P450 enzyme
MeO
HO
HO

NMe
H

MeO

(R)-reticuline

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Phytochem 1993 32 79

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Tetrahedron Lett 1994 35 3897

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis

H3CO
HO
HO
H3CO

NCH3
H

(S)-reticuline

Trends in Biotechnology (2005), 23(7), 331-333.

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis
3400 BC
joy plant by Sumerians

O
O

O
H

1895 Heinrich Dreser working for The Bayer Company

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CO2H
OH

HO

NMe

O
O

HO

H
O

morphine

NMe

heroin

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CO2H
O
O

salicylate

aspirin

S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinoline Biosynthesis
Immunolocalization used to track location of enzymes in morphine biosynthesis

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S. OConnor
Biochemistry 704
2/5/08

Tetrahydroisoquinolin
e Biosynthesis

Immunolocalization used to track

location of enzymes in morphine
biosynthesis

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TIFF (Uncompressed) decompressor
are needed to see this picture.

Curr Opin Plant Biol 2005 8 292

S. OConnor
Biochemistry 704
2/5/08

Human Morphine
Biosynthesis
we have an opiate receptor
thought to bind peptides
YPFP (Science 1981 212 75)

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PNAS 2004 101 14091

PNAS 2005 102 8495

S. OConnor
Biochemistry 704
2/5/08

Caffeine Biosynthesis
Purine
xanthosine

7-methylxanthosine

O
N

HN
O

HN

N O

N
H

HO

OH

OH

N
H

7-methylxanthine

CH3
N

N O
HO

HN

OH

N
H

CH3
N
N

OH

theobromine
O
HN
O

CH3
N

N
N
CH3

Caffeine
O
H 3C
O

CH3
N

N
N
CH3

NH2

HN
N

N O
HO

OPO3
OH

N O
HO

OPO3
OH

O
N

HN
H2N

N O
HO

OPO3
OH

S. OConnor
Biochemistry 704
2/5/08

Caffeine Biosynthesis
Purine

Trends Plant Sci. (2001) 6 407.

S. OConnor
Biochemistry 704
2/5/08

Caffeine Biosynthesis
Engineering
xanthosine

7-methylxanthosine

O
N

HN
O

N
H

HN

N O
HO

OH

OH

N
H

7-methylxanthine

CH3
N

N O
HO

OH

HN
O

N
H

CH3
N
N

theobromine
O
HN
O

OH

CH3
N

N
N
CH3

Caffeine
O
H 3C
O

CH3
N

N
N
CH3

Genetically engineered decaf coffee

70% reduction in caffeine content by using RNAi of theobromine synthase
otherwise normal phenotype
Nature (2003) 423, 823
Caffeine appears to act as insecticide
Trans. Res 2006
Recently discovered natural variant deficient in caffeine synthase
Nature (2004) 429, 826.
Promoter of the 3 methyl transferases recently discovered
J. Biotech. (2005) 119, 20-25.

S. OConnor
Biochemistry 704
2/5/08

Ergot Alkaloids
tryptophan

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Plant Med 2006

S. OConnor
Biochemistry 704
2/5/08

Ergot Alkaloids
tryptophan

O
O
O P O P OH
O
O

HO

NMe

CO2H
NH2

HN

CO2H
H2N

HN

N
H

OH
H
NMe
H

HN

CO2H
MeHN

OH

OH

NMe

NMe

HN

HN

HN

CO2H
MeHN

HN

NMe

NMe

HN

HN

Mol. Gen. Gen. (1999) 261, 133-141

The Alkaloids, G. Cordell pp. 170-218
S. OConnor
Biochemistry 704
2/5/08

Ergot Alkaloids
tryptophan

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Mol. Gen. Gen. (1999) 261, 133-141

The Alkaloids, G. Cordell pp. 170-218

S. OConnor
Biochemistry 704
2/5/08

Ergot Alkaloids
tryptophan
O
O

OH
H

NH

HN

NMe
H
HN

NMe

HN

HN

NMe

N
HO
O

Ergovaline eliminated from a strain

of symbiant
ryegrass staggers
(PNAS (2001) 98 12820)

O
O

NMe

HN

HO
O
NH

NH

Journal of Biological Chemistry (1995), 270(41), 24475-81.

O
O

NMe

HN

NH

HN

NMe

Mol. Gen. Gen. (1999) 261, 133-141

The Alkaloids, G. Cordell pp. 170-218

NH

HN

Potential for combinatorial biosynthesis?

Central amino acid varies

S
O

HN

NMe

NH

NMe

HN

S. OConnor
Biochemistry 704
2/5/08

Ergot Alkaloids
tryptophan

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Mol. Gen. Gen. (1999) 261, 133-141

The Alkaloids, G. Cordell pp.
S.170-218
OConnor
Biochemistry 704
2/5/08

Terpene Indole Alkaloids

tryptophan

COOH

TDC

NH2

NH2

N
H

N
H

tryptamine

STR
H

non mevalonate
terpene biosynthesis

OH
O

O
MeO2C

MeO2C

cathenamine

O HO

OH

strictosidine

OH

SGS

N
O

OH

OH

N
H

OH

MeO2C

O
HO

secologanin

N
H

N
H

NH

N
H

NH

MeO2C

N
H
OH

4,21-dehydrogeissoschizine

O
OH

MeO2C

S. OConnor
Biochemistry 704
2/5/08

Terpene Indole Alkaloids

tryptophan

N
H

MeO2C

CHO

SB

N
H

N H
H

MeO2C

OH

4,21dehydrogeissoschizine

CO2Me

OH

polyneuridine aldehyde

4,5dehydrogeissoschizine

PNAE
AcO

OH

AcO

VR

N H
H

CHO

OH

AcO

VH

VS

N H
H

vomilenine

1,2-dihydrovomilenine

epi-vellosimine

vinorine

DHVR
AcO

OH
N

N H
H

acetylnorajmaline

AAE

HO

OH
N

N H
H

norajmaline

NMT

OH

HO

N H
Me

ajmaline

S. OConnor
Biochemistry 704
2/5/08

Terpene Indole Alkaloids

tryptophan
here for sarpagine
2

N
H

5
7

10

8 13

MeO2C

15

11
12
14

3
5
7

OHC
MeO2C 13
1

10

8 13

MeO2C

15

11

N
12

5
7

10

12

5 7

11

10

14

13

9
8

CHO
CO2Me

4,21-dihydrogeissoschizine
(deglycosylated strictosidine)
corynanthe type
3

5 7

N
H

10

13

5 7

CH2OH
CO2Me

10

13

9
8

CH2OH
CO2Me

preakuammicine
strychnos type

S. OConnor
Biochemistry 704
2/5/08

Terpene Indole Alkaloids

tryptophan
2

N
H

5
7

10

8 13

MeO2C

15

11
12
14

OH

4,21-dihydrogeissoschizine
(deglycosylated strictosidine)
corynanthe type

N
H

10

5 7
6

13

5 7

CH2OH
CO2Me

10

N
H

CH2OH
CO2Me

preakuammicine
strychnos type

11

5 7

13

10

CH2OH
CO2Me

11
9

5 7

8
14

13

12

13

N
H

12

OH

CO2Me

stemmadenine

5
3

N H

2
1

N
H

7
8

12

10 9

11
13 14

CO2Me

tabersonine
aspidosperma type

N 10

8
9
11 12

2
1

N
H

CO2Me

3
2
1

N
H

14
13 10

8
9
11

CO2Me

catharanthine
iboga type

13

12

14

CO2Me

dehydrosecodine

5
7

9 11

N
H

14
13

10

3
14

12

N
H

13

N
10

7
9
11

CO2Me

12

S. OConnor
Biochemistry 704
2/5/08