Encyclopedia_of_Chemical_Biology_(Wiley,_2008).pdf

Alkaloid Biosynthesis

Sarah E. O’Connor, Department of Chemistry, Massachusetts Institute of

Technology, Cambridge, Massachusetts

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Article Contents

•

Biologic Background

•

Isoquinoline Alkaloids

doi: 10.1002/9780470048672.wecb004

•

Terpenoid Indole Alkaloids

•

Tropane Alkaloids

How nature synthesizes complex secondary metabolites, or natural

products, can be studied only by working within the disciplines of both

chemistry and biology. Alkaloids are a complex group of natural products

with diverse mechanisms of biosynthesis. This article highlights the

biosynthesis of four major classes of plant-derived alkaloids. Only plant

alkaloids for which signiﬁcant genetic information has been obtained were

chosen for review. Isoquinoline alkaloid, terpenoid indole alkaloid, tropane

alkaloid, and purine alkaloid biosynthesis are described here. The article is

intended to provide an overview of the basic mechanism of biosynthesis for

selected members of each pathway. Manipulation of these pathways by

metabolic engineering is highlighted also.

•

Purine Alkaloids

Alkaloids are a highly diverse group of natural products re-

lated only by the presence of a basic nitrogen atom located

at some position in the molecule. Even among biosynthetically

related classes of alkaloids, the chemical structures are often

highly divergent. Although some classes of natural products

have a recognizable biochemical paradigm that is centrally ap-

plied throughout the pathway, for example, the “assembly line”

logic of polyketide biosynthesis (1), the biosynthetic pathways

of alkaloids are as diverse as the structures. It is difﬁcult to

predict the biochemistry of a given alkaloid based solely on

precedent, which makes alkaloid biosynthesis a challenging, but

rewarding, area of study.

have been cloned successfully, and many more enzymes have

been puriﬁed from alkaloid-producing plants or cell lines (4–6).

Identiﬁcation and study of the biosynthetic enzymes has a sig-

niﬁcant impact on the understanding of the biochemistry of the

pathway. Furthermore, genetic information also can be used to

understand the complicated localization patterns and regulation

of plant pathways. This article focuses on the biochemistry re-

sponsible for the construction of plant alkaloids and summarizes

the biosynthetic genes that have been identiﬁed to date. Some of

these pathways have been the subject of metabolic engineering

studies; the results of these studies are mentioned here also. An

excellent, more detailed review that covers the biochemistry and

genetics of plant alkaloid biosynthesis up until the late 1990s is

available also (7).

Biologic Background

Hundreds of alkaloid biosynthetic pathways have been studied

by chemical strategies, such as isotopic labeling experiments

(2, 3). However, modern molecular biology and genetic method-

ologies have facilitated the identiﬁcation of alkaloid biosyn-

thetic enzymes. This article focuses on pathways for which a

signiﬁcant amount of genetic and enzymatic information has

been obtained. Although alkaloid natural products are produced

by insects, plants, fungi, and bacteria, this article focuses on

four major classes of plant alkaloids: the isoquinoline alkaloids,

the terpenoid indole alkaloids, the tropane alkaloids, and the

purine alkaloids.

In general, plant biosynthetic pathways are understood poorly

when compared with prokaryotic and fungal metabolic path-

ways. A major reason for this poor understanding is that genes

that express complete plant pathways typically are not clus-

tered together on the genome. Therefore, each plant enzyme

often is isolated individually and cloned independently. How-

ever, several enzymes involved in plant alkaloid biosynthesis

Isoquinoline Alkaloids

The isoquinoline alkaloids include the analgesics morphine

and codeine as well as the antibiotic berberine ( Fig. 1a ).

Morphine and codeine are two of the most important analgesics

used in medicine, and plants remain the main commercial

source of the alkaloids (8). Development of plant cell cultures

of Eschscholzia californica , Papaver somniferum ,and Coptis

japonica has aided in the isolation and cloning of many enzymes

involved in the biosynthesis of isoquinoline alkaloids (9).

Early steps of isoquinoline biosynthesis

Isoquinoline biosynthesis begins with the substrates dopamine

and p -hydroxyphenylacetaldehyde ( Fig. 1b ). Dopamine is made

from tyrosine by hydroxylation and decarboxylation. Enzymes

that catalyze the hydroxylation and decarboxylation steps in

either order exist in the plant, and the predominant pathway

Alkaloid Biosynthesis

(a)

(b)

Figure 1 (a) Representative isoquinoline alkaloids. (b) Early biosynthetic steps of the isoquinoline pathway yield the biosynthetic intermediate

(S)-reticuline, the central biosynthetic intermediate for all isoquinoline alkaloids. (c) Berberine and sanguinarine biosynthesis pathways. (d)Morphine

biosynthesis. NCS, norcoclaurine synthase; 6-OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase (Cyp80B); NMTC,

N-methylcoclaurine 3 -hydroxylase; 4 -OMT, 3 -hydroxy-N-methylcoclaurine 4 -O-methyltransferase; BBE, berberine bridge enzyme; SOMT, scoulerine

9-O-methyltransferase; CS, canadine synthase; TBO, tetrahydroprotoberberine oxidase; CHS, cheilanthifoline synthase; SYS, stylopine synthase; NMT,

N-methyltransferase; NMSH, N-methylstylopine hydroxylase; P6H protopine 6-hydroxylase; DHPO, dihydrobenzophenanthridine oxidase; RO, reticuline

oxidase; DHR, dihydroreticulinium ion reductase; STS, salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol acetyltransferase; COR,

codeinone reductase.

for formation of dopamine from tyrosine is not clear. The

second substrate, p -hydroxyphenylacetaldehyde, is generated by

transamination and decarboxylation of tyrosine (10, 11).

Condensation of dopamine and p -hydroxyphenylacetaldehyde

is catalyzed by norcoclaurine synthase to form (S)-norcoclaurine

( Fig. 1b ). Two norcoclaurine synthases with completely unre-

lated sequences were cloned ( Thalictrum ﬂavum and C. japon-

ica ) and heterologously expressed in E. coli (12–14). One is ho-

mologous to iron-dependent diooxygenases, whereas the other

is homologous to a pathogenesis-related protein. Undoubtedly,

future experiments will shed light on the mechanism of these

enzymes and on how two such widely divergent sequences can

catalyze the same reaction.

One of the hydroxyl groups of (S)-norcoclaurine is methy-

lated by a S-adenosyl methionine-(SAM)-dependent O-methyl

transferase to yield (S)-coclaurine. This enzyme has been

cloned, and the heterologously expressed enzyme exhibited

the expected activity (15–17). The resulting intermediate is

Alkaloid Biosynthesis

(c)

(d)

Figure 1 ( continued )

Alkaloid Biosynthesis

then N-methylated to yield N-methylcoclaurine, an enzyme

that has been cloned recently (18, 19). N-methylcoclaurine, in

turn, is hydroxylated by a P450-dependent enzyme (CYP80B),

N-methylcoclaurine 3 -hydroxylase, that has been cloned (20,

21). The 4 hydroxyl group then is methylated by the

enzyme 3 -hydroxy-N-methylcoclaurine 4 -O-methyltransferase

(4 -OMT) to yield (S)-reticuline, the common biosynthetic inter-

mediate for the berberine, benzo(c)phenanthridine, and morphi-

nan alkaloids ( Fig. 1b ). The gene for this methyl transferase also

has been identiﬁed (15, 22). These gene sequences also were

used to identify the corresponding T. ﬂavum genes that encode

the biosynthetic enzymes for reticuline from a cDNA library

(23). At this point, the biosynthetic pathway then branches to

yield the different structural classes of isoquinoline alkaloids.

P450 enzyme, protopine 6-hydroxylase, to yield an intermediate

that rearranges to dihydrosanguinarine (40). This intermediate

also serves as the precursor to the benzo(c)phenanthridine alka-

loid macarpine ( Fig. 1a ). The copper-dependent oxidase dihy-

drobenzophenanthridine oxidase, which has been puriﬁed (41,

42), then catalyzes the formation of sanguinarine from dihy-

drosanguinarine.

Additional enzymes from other benzo(c)phenanthidine alka-

loids have been cloned. For example, an O-methyl transferase

implicated in palmitine biosynthesis has been cloned recently

(43).

Morphine biosynthesis

Berberine biosynthesis

The later steps of morphine biosynthesis have been inves-

tigated in P. somniferum cells and tissue. Notably, in mor-

phine biosynthesis, (S)-reticuline is converted to (R)-reticuline,

thereby epimerizing the stereocenter generated by norcoclaurine

synthase at the start of the pathway ( Fig. 1d ). (S)-reticuline is

converted to (R)-reticuline through a 1,2-dehydroreticuline in-

termediate. Dehydroreticuline synthase catalyzes the oxidation

of (S)-reticuline to 1,2-dehydroreticulinium ion (44). This en-

zyme has not been cloned but has been puriﬁed partially and

shown to be membrane-associated. This intermediate then is

reduced by dehydroreticuline reductase, an NADPH-dependent

enzyme that stereoselectively transfers a hydride to dehydroreti-

culinium ion to yield (R)-reticuline. This enzyme has not been

cloned yet but has been puriﬁed to homogeneity (45).

Next, the key carbon–carbon bond of the morphinan alka-

loids is formed by the cytochrome P450 enzyme salutaridine

synthase. Activity for this enzyme has been detected in microso-

mal preparations, but the sequence has not been identiﬁed (46).

The keto moiety of the resulting product, salutaridine, then is

stereoselectively reduced by the NADPH-dependent salutaridine

reductase to form salutardinol. The enzyme has been puriﬁed

(47), and a recent transcript analysis proﬁle of P. sominiferum

has resulted in the identiﬁcation of the clone (48). Salutaridinol

acetyltransferase, also cloned, then transfers an acyl group from

acetyl-CoA to the newly formed hydroxyl group, which results

in the formation of salutaridinol-7-O-acetate (49). This modiﬁ-

cation sets up the molecule to undergo a spontaneous reaction

in which the acetate can act as a leaving group. The resulting

product, thebaine, then is demethylated by an as yet uncharac-

terized enzyme to yield neopinione, which exists in equilibrium

with its tautomer codeinone. The NADPH-dependent codeinone

reductase catalyzes the reduction of codeinone to codeine and

has been cloned (50, 51). Finally, codeine is demethylated by

an uncharacterized enzyme to yield morphine.

The localization of isoquinoline biosynthesis has been in-

vestigated at the cellular level in intact poppy plants by using

in situ RNA hybridization and immunoﬂouresence microscopy.

The localization of 4 -OMT (reticuline biosynthesis), berberine

bridge enzyme (saguinarine biosynthesis), salutaridinol acetyl-

transferase (morphine biosynthesis), and codeinone reductase

(morphine biosynthesis) has been probed. 4 -OMT and salu-

taridinol acetyltransferase are localized to parenchyma cells,

whereas codeinone reductase is localized to laticifer cells in

(S)-reticuline is converted to (S)-scoulerine by the action

of a well-characterized ﬂavin-dependent enzyme, berberine

bridge enzyme ( Fig. 1c ). This enzyme has been cloned from

several plant species, and the mechanism of this enzyme

has been studied extensively (24–28). (S)-scolerine is then

O-methylated by scoulerine 9-O-methyltransferase to yield

(S)-tetrahydrocolumbamine. Heterologous expression of this

gene in E. coli yielded an enzyme that had the expected

substrate speciﬁcity (29). A variety of O-methyl transferases

also have been cloned from Thalictrum tuberosum (30). The

substrate-speciﬁc cytochrome P450 oxidase canadine synthase

(31) that generates the methylene dioxy bridge of (S)-canadine

has been cloned recently (32). The ﬁnal step of berberine

biosynthesis is catalyzed by a substrate-speciﬁc oxidase, tetrahy-

droprotoberberine oxidase, the sequence of which has not been

identiﬁed yet (33).

Overproduction of berberine in C. japonica cell suspension

cultures was achieved by selection of a high-producing cell

line (34) with reported productivity of berberine reaching 7 g/L

(35). This overproduction is one of the ﬁrst demonstrations of

production of a benzylisoquinoline alkaloid in cell culture at

levels necessary for economic production. This cell line has

facilitated greatly the identiﬁcation of the biosynthetic enzymes.

Sanginarine biosynthesis

The biosynthesis of the highly oxidized benzo(c)phenanthidine

alkaloid sanguinarine is produced in a variety of plants and

competes with morphine production in opium poppy. The path-

way to sanguinarine has been elucidated at the enzymatic

level ( Fig. 1c ) (36). Sanguinarine biosynthesis starts from

(S)-scoulerine, as in berberine biosynthesis. Methylenedioxy

bridge formation then is catalyzed by the P450 cheilanthifo-

line synthase to yield cheilanthifoline (37). A second P450

enzyme, stylopine synthase, catalyzes the formation of the sec-

ond methyenedioxy bridge of stylopine (37). Stylopine syn-

thase from E. californica has been cloned recently (38). Sty-

lopine then is N-methylated by (S)-tetrahydroprotoberberine

cis -N-methyltransferase to yield (S)- cis -N-methylstylopine, an

enzyme that has been cloned recently from opium poppy (39).

A third P450 enzyme, (S)- cis -N-methylstylopine hydroxylase,

then forms protopine. Protopine is hydroxylated by a fourth

Alkaloid Biosynthesis

sections of capsule (fruit) and stem from poppy plants. Berber-

ine bridge enzyme is found in parenchyma cells in roots. There-

fore, this study suggests that two cell types are involved in iso-

quinoline biosynthesis in poppy and that intercellular transport

is required for isoquinoline alkaloid biosynthesis (52). Another

study, however, implicates a single cell type (sieve elements and

their companion cells) in isoquinoline alkaloid biosynthesis (53,

54). Therefore, it is not clear whether transport of pathway in-

termediates is required for alkaloid biosynthesis or whether the

entire pathway can be performed in one cell type. Localization

of enzymes in alkaloid biosynthesis is difﬁcult, and, undoubt-

edly, future studies will provide more insight into the trafﬁcking

involved in plant secondary metabolism.

Early steps of terpenoid indole alkaloid

biosynthesis

All terpenoid indole alkaloids are derived from tryptophan and

the iridoid terpene secologanin ( Fig. 2b ). Tryptophan decar-

boxylase, a pyridoxal-dependent enzyme, converts tryptophan

to tryptamine (62, 63). The enzyme strictosidine synthase cat-

alyzes a stereoselective Pictet–Spengler condensation between

tryptamine and secologanin to yield strictosidine. Strictosidine

synthase (64) has been cloned from the plants C. roseus (65),

Rauwolﬁa serpentine (66), and, recently, Ophiorrhiza pumila

(67). A crystal structure of strictosidine synthase from R. ser-

pentina has been reported (68, 69), and the substrate speciﬁcity

of the enzyme can be modulated (70).

Strictosidine then is deglycosylated by a dedicated

β -glucosidase, which converts it to a reactive hemiacetal inter-

mediate (71–73). This hemiacetal opens to form a dialdehyde

intermediate, which then forms dehydrogeissoschizine. The enol

form of dehydrogeissoschizine undergoes 1,4 conjugate addition

to produce the heteroyohimbine cathenamine (74–76). A variety

of rearrangements subsequently act on deglycosylated strictosi-

dine to yield a diversity of indole alkaloid products (77).

Metabolic engineering of morphine

biosynthesis

In attempts to accumulate thebaine and decrease produc-

tion of morphine (a precursor to the recreational drug hero-

ine), codeinone reductase in opium poppy plant was down-

regulated by using RNAi (8). Silencing of codeinone re-

ductase results in the accumulation of (S)-reticuline but not

the substrate codeinone or other compounds on the pathway

from (S)-reticuline to codeine. However, the overexpression

of codeinone reductase in opium poppy plants did result, in

fact, in an increase in morphine and other morphinan alkaloids,

such as morphine, codeine, and thebaine, compared with control

plants (55). Gene expression levels in low morphine-producing

poppy plants have been analyzed also (56). Silencing of berber-

ine bridge enzyme in opium poppy plants also resulted in a

change in alkaloid proﬁle in the plant latex (57).

The cytochrome P450 responsible for the oxidation of

(S)-N-methylcoclaurine to (S)-3 -hydroxy-N-methylcocluarine

has been overexpressed in opium poppy plants, and morphi-

nan alkaloid production in the latex is increased subsequently

to 4.5 times the level in wild-type plants (58). Additionally,

suppression of this enzyme resulted in a decrease in morphi-

nan alkaloids to 16% of the wild-type level. Notably, analysis

of a variety of biosynthetic gene transcript levels in these ex-

periments supports the hypothesis that this P450 enzyme plays

a regulatory role in the biosynthesis of benzylisoquinoline al-

kaloids. Collectively, these studies highlight that the complex

metabolic networks found in plants are not redirected easily or

predictably in all cases.

Ajmaline biosynthesis

The biosynthetic pathway for ajmaline in R. serpentina is one

of the best-characterized terpenoid indole alkaloid pathways.

Much of this progress has been detailed in a recent extensive

review (78). Like all other terpenoid indole alkaloids, ajmaline,

an antiarrhythmic drug with potent sodium channel-blocking

properties (79), is derived from deglycosylated strictosidine

( Fig. 2c ).

A membrane–protein fraction of an R. serpentina extract

transforms labeled strictosidine (80, 81) into sarpagan-type alka-

loids. The enzyme activity is dependent on NADPH and molec-

ular oxygen, which suggests that sarpagan bridge enzyme may

be a cytochrome P450 enzyme. Polyneuridine aldehyde esterase

hydrolyzes the polyneuridine aldehyde methyl ester, which gen-

erates an acid that decarboxylates to yield epi-vellosamine.

This enzyme has been cloned from a Rauwolﬁa cDNA library,

heterologously expressed in E. coli , and subjected to detailed

mechanistic studies (82, 83).

In the next step of the ajmaline pathway, vinorine synthase

transforms the sarpagan alkaloid epi-vellosamine to the ajmalan

alkaloid vinorine (84). Vinorine synthase also has been puriﬁed

from Rauwolﬁa cell culture, subjected to protein sequencing,

and cloned from a cDNA library (85, 86). The enzyme, which

seems to be an acetyl transferase homolog, has been expressed

heterologously in E. coli . Crystallization and site-directed muta-

genesis studies of this protein have led to a proposed mechanism

(87).

Vinorine hydroxylase hydroxylates vinorine to form vom-

ilene (88). Vinorine hydroxylase seems to be a P450 en-

zyme that requires an NADPH-dependent reductase. This

enzyme is labile and has not been cloned yet. Next, the

indolenine bond is reduced by an NADPH-dependent re-

ductase to yield 1,2-dihydrovomilenene. A second enzyme,

1,2-dihydrovomilenene reductase, then reduces this product to

Terpenoid Indole Alkaloids

The terpenoid indole alkaloids have a variety of chemical struc-

tures and a wealth of biologic activities ( Fig. 2a ) (59, 60).

Terpenoid indole alkaloids are used as anticancer, antimalarial,

and antiarrhythmic agents. Although many biosynthetic genes

from this pathway remain unidentiﬁed, recent studies have cor-

related terpenoid indole alkaloid production with the transcript

proﬁles of Catharanthus roseus cell cultures (61).

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