Abstract The biosynthetic pathway leading to

the monoterpenoid indole alkaloid ajmaline in

Rauvolfia serpentiin serpentina is one of the

most studied in the field of natural product

biosynthesis. Ajmaline has a complex structure

which is based on a six-membered ring system

harbouring nine chiral carbon atoms. There are

about fifteen enzymes involved, including some

involving the side reactions of the ajmaline

biosynthetic pathway. All enzymes exhibit pro-

nounced substrate specificity. In the recent years

isolation and sequencing of their cDNAs has

allowed a detailed sequence analysis and com-

parison with functionally related and occasion-

ally un-related enzymes. Site-directed mutations

of several of the ajmaline-synthesizing enzymes

have been performed and their catalytic resi-

dues have been identified. Success with over-

expression of the enzymes was an important

step for their crystallization and structural

analysis by X-ray crystallography. Crystals with

sufficient resolution were obtained from the

major enzymes of the pathway. Strictosidine

synthase has a 3D-structure with a six-bladed b-

propeller fold the first time such a fold found in

the plant kingdom. Its ligand complexes with

tryptamine and secologanin, as well as struc-

ture-based sequence alignment, indicate a pos-

sible evolutionary relationship to several

primary sequence-unrelated structures with this

fold. The structure of strictosidine glucosidase

was determined and its structure has as a (b/a)8

barrel fold. Vinorine synthase provides the first

3D structure of a member of BAHD enzyme

super-family. Raucaffricine glucosidase involved

in a side-route of ajmaline biosynthesis has been

crystallized. The ajmaline biosynthetic pathway

is an outstanding example where many enzymes

3D-structure have been known and where there

is a real potential for protein engineering to

yield new alkaloid.

Keywords Rauvolfia alkaloids Æ Strictosidine

synthase Æ Strictosidine glucosidase Æ Vinorine

synthase Æ 3D-structures Æ X-ray crystallography

J. Stockigt (&) Æ M. Ruppert Æ L. Barleben Æ X. Ma ÆE. Loris Æ M. HillDepartment of Pharmaceutical Biology, Institute ofPharmacy, Johannes Gutenberg-Universitat,Staudinger Weg 5, D-55099 Mainz, Germanye-mail: stoeckig@mail.uni-mainz.de

S. PanjikarEuropean Molecular Biology Laboratory HamburgOutstation Deutsches Elektronen-Synchrotron,Notkestrasse 85, D-22603 Hamburg, Germany

J. StockigtCollege of Pharmaceutical Sciences, ZhejiangUniversity, 353 Yan An Road, Hangzhou 310031,China

Phytochem Rev (2007) 6:15–34

DOI 10.1007/s11101-006-9016-2

123

The molecular architecture of major enzymesfrom ajmaline biosynthetic pathway

Joachim Stockigt Æ Santosh Panjikar ÆMartin Ruppert Æ Leif Barleben Æ Xueyan Ma ÆElke Loris Æ Marco Hill

Received: 10 May 2006 / Accepted: 8 July 2006 / Published online: 27 February 2007� Springer Science+Business Media B.V. 2007

Introduction

During the last decade, research in the field of

alkaloid biosynthesis has been growing steadily.

The progress is mainly based on the systematic

application of efficient biological systems, such as

plant cell suspension cultures, on the implemen-

tation of biochemical methodologies, for example

experiments focused on enzyme isolation and

characterization and on the application of

molecular biology and genetic approaches, such

as isolating the cDNAs coding for enzymes in-

volved in long and complex biosynthetic path-

ways. All these methodologies were helpful in

order to explore details of alkaloid biosynthesis in

the Indian plant Rauvolfia serpentina Benth ex.

Kurz, already used about 2500 years ago in Ay-

urvedic medicine.

The aim of our research on Rauvolfia is to

elucidate not only the mechanism of each enzyme

involved in the multi-step pathway leading to the

anti-arrhythmic alkaloid ajmaline but also to

investigate the complete enzymatic scenario at

the molecular level which is a challenging task

because of the structural complexity of this alka-

loid (Fig. 1). The structural complexity is bio-

synthetically built up by a great number of

different enzymes of various classes. The ajmaline

structure consists of a six-membered ring system

and in addition to the chiral Nb nitrogen the

alkaloid harbors nine additional asymmetric car-

bon atoms at the positions 2, 3, 5, 7, 15, 16, 17, 20,

21 (see ajmaline in Fig. 1).

Detection, isolation and characterization of

these enzymes in Rauvolfia cell culture was

investigated allowing a detail description of each

step of the pathway including the putative sub-

strates and products of each enzyme (Ruppert

et al. 2005).

Although there existed some general knowl-

edge of ajmaline biosynthesis, the analysis of a

pure enzyme in term of biochemical properties

and primary structure was not previously possi-

ble. The application of the molecular genetics and

molecular biology has changed the situation. The

cDNAs of soluble enzymes of the pathway can

now be isolated and functionally expressed using

the ‘‘reverse genetic approach’’ starting with

partial amino acid sequences obtained from

purified Rauvolfia enzymes after partial digestion

and sequencing of the resulting peptides.

The overexpression of several Rauvolfia pro-

teins enabled us to produce enzymes in a highly

purified form and most of them were suitable for

crystallizations and the diffracting crystals could

be used for X-ray analysis in order to determine

their 3D-structure and to explore mechanistic and

evolutionary aspects of these catalysts. Crystalli-

zation of enzymes is not in general an easy task

requiring many crystallization trials and mg

amounts of protein to achieve a final crystalliza-

tion condition and further to optimize the crys-

tallization conditions. One of our major aims is to

investigate structurally the majority of the en-

zymes we have detected in the pathway leading to

the Rauvolfia alkaloid ajmaline. This aim would

previously have been difficult because the amount

of pure enzymes available from natural plant

biosynthesis are low and the success rate of

solving a 3D-structure of protein is, even for

soluble proteins, modest. A recent study by De-

Lucas et al. (2005) summarized crystallization

results from the NIH Structural Genomics Ini-

tiative. The analysis of seven NIH structural ge-

nomics centers in USA revealed that the success

rates for 3D-structure determination is around

5%, although most of the proteins came from

prokaryotic sources. There seems to be two major

‘‘bottlenecks’’. Firstly the success of producing

soluble proteins is low, since only 1/3 of cloned

cDNAs gave enough soluble protein to proceed to

crystallization. Secondly only 23% of the solubly

expressed proteins have been crystallized. Success

rates for proteins from eukaryotic systems would

be expected to be much lower. In spite of the fact

that sophisticated instruments for high-throughput

crystallization have been developed which can

carry out thousands of crystallization experiments

(Krupka et al. 2002) using small amounts of pro-

tein. The limitation is ability to obtain well-ordered

crystals for X-ray analysis. Once X-ray data is

collected and processed the structure determina-

tion is usually successful. In recent years, speed of

structure determination has been enhanced by the

development of advanced crystallographic soft-

ware and assembling these programmes into a so-

called pipeline for crystallography i.e. structure

determination. An example is Auto-Rickshaw the

16 Phytochem Rev (2007) 6:15–34

123

automatic crystal structure determination platform

used at the EMBL Hamburg Outstation (Panjikar

et al. 2005). It is used at protein crystallography

beamlines for fast validation of the X-ray diffrac-

tion experiment while the crystal is still in the beam

or near the beamline. It can ensure that the X-ray

data of sufficient quality and quantity for successful

structure determination at latter stage. Crystallo-

graphic refinement is not yet the part of the plat-

form therefore after the initial model building, user

intervention is necessary to complete the model

building and refinement. This pipeline has been

instrumental in solving the structure of enzymes

from the ajmaline biosynthetic pathway.

The present article summarizes the results of

the 3D-structure determination of major enzymes

of the ajmaline biosynthetic pathway as well as

some substrate complexes.

The ajmaline pathway is summarized in the

Fig. 1. The pathway is initiated by the condensa-

tion of the biosynthetic precursors—tryptamine

and secologanin a process catalyzed by strict-

osidine synthase (STR1). Tryptamine is an

b-arylethylamine whereas secologanin is a mon-

oterpenoid aldehyde. Such a condensation of an

amine and aldehyde is called a Pictet–Spengler

type reaction (Pictet and Spengler 1911), it is

completely stereo-selective and functions as the

Fig. 1 Enzymatic biosynthesis of the antiarrhythmicindole alkaloid ajmaline in Rauvolfia serpentina cellsuspensions including some side reactions of the pathway.STR1 = Strictosidine synthase (EC 4.3.3.2), SG = Strict-osidine glucosidase (EC 3.2.1.105), SBE = Sarpaganbridge Enzyme, PNAE = Polyneuridine aldehyde esterase(EC 3.1.1.78), VS = Vinorine Synthase (EC 2.3.1.160),VH = Vinorine hydroxylase (EC 1.14.13.75), CPR = Cy-tochromeP450 reductase, VR = Vomilenine reductase

(EC 1.5.1.32), DHVR = 1,2-Dihydrovomilenine reductase(EC 1.5.1.73), AAE = Acetylajmalan esterase,NAMT = Norajmalan methyltransferase, PR = Perakinereductase, RG = Raucaffricine glucosidase (EC 3.2.1.125).Abbreviations in green letters means that the appropriatecDNA has been heterologously overexpressed and thedotted line indicates the side routes of the pathway. Chiralcarbon atoms of six-membered structure of the monoterp-enoid indole alkaloid ajmaline are numbered

Phytochem Rev (2007) 6:15–34 17

123

biosynthetic entry to around 2000 monoterpenoid

indole alkaloids in higher plants. Of these alka-

loids several have medical and pharmacological

applications. Examples are antihypertensive

compounds ajmalicine and reserpine, the anti-

cancer compounds vinblastine, vincristine, cam-

ptothecin, the Vinca alkaloid vincamine used

against disturbed cerebral blood flow or the

antiarrhythmic drug (Creasey 1994) discussed

here. In conclusion, STR1 and the enzyme which

follows STR1 in the pathway, strictosidine glu-

cosidase (SG), occupy key positions in all the

pathways leading to all these therapeutic agents.

The third enzyme described here in detail, vino-

rine synthase (VS), is responsible for generation

of the basic carbon skeleton of the target com-

pound ajmaline.

Strictosidine synthase (STR1) crystallization

and X-ray analysis

The cDNA of this extraordinary enzyme was

cloned from R. serpentina first such example in

the entire field of alkaloid biosynthesis (Kutchan

1988; Bracher and Kutchan 1992). Later on, the

primary structure of the STR1s from two other

sources, Catharanthus roseus G. Don (DeWaal

et al. 1995) and Ophiorrhiza pumila Champ.

(Yamazaki et al. 2003) were obtained. The en-

zyme has been reviewed several times during the

last one and a half decades (Kutchan 1993; Rup-

pert and Stockigt 1999) resulting in STR1 being

one of the best described enzymes of alkaloid

metabolism in general and Rauvolfia in particular.

STR1 was overexpressed in Escherichia coli.

The enzyme was purified to high purity and on

the mg-scale using nickel-NTA affinity chroma-

tography. Enzymatic digestion then delivered the

His6-tag-free enzyme which could be crystallized

after performing a broad screening of crystalli-

zation conditions. The best crystals were grown

using the hanging-drop vapor diffusion method as

described recently (Ma et al. 2004a; Koepke et al.

2005). Crystals were cryoprotected with glycerol

before being flash frozen at 100 K temperature

and the X-ray diffraction data were collected on a

synchrotron beam line. This methodology was the

same for most of the other Rauvolfia enzymes we

have crystallized and those which are described in

this article. Crystals of STR1 (Fig. 2a) belong to

space group R3. There were no protein structures

available showing significant sequence homology

to STR1 and consequently the enzyme was la-

beled with seleno-methionine in order to use the

multiple wavelength anomalous dispersion

(MAD) approach to obtain experimental phases.

The structure was solved using Auto-Rickshaw

pipeline (Panjikar et al. 2005) as described in (Ma

et al. 2006) and refined to 2.4 A resolution.

Overall structure of STR1

The overall architecture of STR1 has a six-bladed

b-propeller fold (Ma et al. 2006). The b-propeller

architecture which consists of 5 classes, has been

described several times in literature, especially

because of their different functions and their

importance in human diseases (Pons et al. 2003).

In STR1 each blade is formed by four-stranded

anti-parallel b-strands. All blades are arranged

around six-fold axis (Fig. 2b). Such an overall

propeller structure with various numbers of blades

has been found in nature to be relatively common.

But the enzymes with the 6-bladed fold exhibit

completely different catalytic functions (Scharff

Fig. 2 Strictosidine synthase (STR1) (a) Crystals of STR1obtained using the hanging drop vapour diffusion tech-nique. (b) Overall structure of STR1 resembling a six-bladed b-propeller fold, a fold which has been detected forthe first time in the plant kingdom. Four-stranded anti-parallel b-sheets are arranged around one axis forming thecatalytic center in which the Pictet–Spengler condensationbetween tryptamine and secologanin is catalyzed. (c)Tryptamine-binding pocket of STR1 with electron densitymap (Fo – Fc map contoured at 4r) and surroundingamino acid residues. The structure of the complex isobtained from a co-crystallization experiment of STR1 andtryptamine. (d) Secologanin binding-site of STR1 withelectron density map (Fo – Fc map contoured at 4r) andsurrounding amino acid residues. The structure of thecomplex was obtained after soaking of secologanin intoSTR1 crystals. (e) Structure-based sequence alignment ofSTR1 from Rauvolfia cells with other six-bladed b-propeller structures from different origin. Putative cata-lytic residues are marked by a black dot above thesequence and the new detected common motif indicatingevolutionary relationship of these enzymes is indicated byblack triangles (Ma et al. 2006)

c

18 Phytochem Rev (2007) 6:15–34

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Phytochem Rev (2007) 6:15–34 19

123

et al. 2001; Harel et al. 2004) and are not of plant

origin. STR1 is, therefore, the first example of a 6-

bladed b-propeller found in the whole plant

kingdom. It is also the first time that the 3D-

structure of an enzyme catalyzing a Pictet–Spen-

gler reaction, well established in the chemical

synthesis of alkaloids for nearly one hundred years

(Pictet and Spengler 1911, for recent reviews see

Cox and Cook 1995; Chrzanowska and Ro-

zwadowska 2004) has been determined. The

structure of STR1 (Fig. 2b) contains only 3 heli-

ces. Two of them are connected with a disulfide

‘‘bridge’’, which seems to be a common feature in

the STR1 enzyme family. This ‘‘bridge’’ between

Cys89 and Cys101 is conserved in all the known

members of this family and when Cys89 was mu-

tated to Ser the resulting enzyme completely lost

its catalytic activity. From the 3D-structure it can

be deduced that the S–S bond is important for

both the overall architecture and the shape of the

binding pocket indicating it is structurally rather

than functionally important. Two further struc-

tural features are important for the shape of the

binding pocket, a helix between two b-sheets (b-B

and b-C) in blade 3 and a connecting loop between

b-B and b-C in blade 5. Both of them form a cap

over the active site which could influence the entry

of substrate to the site.

Earlier work on STR1 from Catharanthus

indicated the occurrence of several isoforms of

the enzyme in this species (Pfitzner and Zenk

1989; DeWaal et al. 1995), which might be

formed by different glycosylations of the protein.

But in contrast, such isoforms have not been de-

tected in Rauvolfia. Sequence analysis of both

enzymes showed that STR1 from Rauvolfia con-

tains only one putative glycosylation site (Asn91),

which is located on the enzyme surface between

the a-1 and a-2 helices. This site is conserved

throughout all the STR1 family members. STR1

from Catharanthus, however, shows a second

putative glycosylation site (Asn187) which is re-

placed by Asp in Rauvolfia and which is located

on the a-3 helix. The 3D-structure of the Catha-

ranthus enzyme which is not known at the present

time, should now been easier to obtain after the

structure of the Rauvolfia enzyme became avail-

able, as these two enzymes shares high sequence

homology.

Catalytic center and site-directed mutagenesis

of STR1—Ligand complexes with tryptamine

and secologanin

The active site of STR1 is located near the six-

fold symmetry axis. A channel links the binding

pocket on one side of the molecule with the sur-

face of the enzyme. The residues of the catalytic

center are mostly hydrophobic consisting of sev-

eral Phe, Val, Tyr and Met residues as well as one

Trp and Leu residue. In contrast, the entrance of

the channel consists of polar amino acids such as

the positively charged His307 and His277 but in

addition exhibits a few hydrophobic residues such

as Met276 and Phe324.

A complex structure of STR1 with the sub-

strate tryptamine (TAM) gives further insight

into the nature of the catalytic site. The STR1-

TAM complex (Fig. 2c) nicely shows that the

substrate is located deep in the pocket. The amino

group of tryptamine is coordinated to Glu309 and

a water molecule (W41) is bound to the indole

nitrogen. The flat indole part of the substrate

stacks between the aromatic rings of Tyr151 and

Phe226 keeping the amine in an optimum posi-

tion to the second substrate secologanin (SEL),

which is situated in the same binding pocket (see

the complex structure STR1-SEL, Fig. 2d). The

aldehyde group of secologanin, which during the

Pictet–Spengler reaction condenses with the

amine function of tryptamine, is very near to the

NH2-group and is coordinated with the same

amino acid (Glu309). The ester group of seco-

loganin points to the bottom of the catalytic

centre whereas the hydrophilic part, the glucose

unit, faces toward the solvent region near the

entrance of the pocket. His307 holds the gluco-

sidic part by coordination of two oxygens with the

nitrogens of His. In fact, there are only three

polar amino acids located in the catalytic centre

near the substrates. These are Glu309, Tyr151 and

His307, respectively (Fig. 2b). Site-directed

mutagenesis of these residues provides a greater

understanding of the reaction and of the impor-

tance of the single residues in the reaction. When

Glu309 was replaced by Ala not much influence

on the Km of tryptamine was observed in contrast

to the turnover rate kcat which greatly decreased

(about 880-fold). The major role of Glu309 is

20 Phytochem Rev (2007) 6:15–34

123

obviously de-protonation of the tryptamine ami-

no function which then condenses with the alde-

hyde function of secologanin which Schiff-base

formation followed by ring-closure and synthesis

of the enzyme product 3a(S)-strictosidine. Glu309

must, therefore, be an essential amino acid for the

catalytic process. Indeed, the Asp/Glu-selective

inhibitor N, N¢-dicyclohexylcarbodiimide (DCC)

knocked out the STR1 activity completely, veri-

fying the catalytic role of Glu309. Displacement

of the other polar residues, Tyr151 and His307, by

Phe and Ala respectively, caused less influence on

enzymatic activity. The Km for tryptamine in-

creased about 3-fold in the first case, however,

there was a 130-fold increase for secologanin in

the second case. As suggested above, His307

plays a significant role in binding secologanin by

keeping the best structural orientation for seco-

loganin in the pocket. At this stage of investiga-

tion it is clear that Glu309 has been identified as

the essential catalytic residue in the binding

pocket of STR1 and is the amino acid necessary

for a Pictet–Spengler reaction (Falbe and Regitz

1991).

As known from earlier work strictosidine

synthase from Catharanthus or Rauvolfia is a

highly substrate specific enzyme (Treimer and

Zenk 1979). It does not, for instance, accept

dopamine, which is a precursor of monoterpe-

noid isoquinoline alkaloids as a substrate or

isoquinolines generated by deacetylipecoside and

norcoclaurine synthase. These later enzymes

should be similar to STR1, especially deacety-

lipecoside and deacetylisoipecoside synthase,

which have been recently detected and described

(De-Eknamkul et al. 1997; De-Eknamkul et al.

2000). Since the amino acid sequence and the

3D-structure of these proteins is not known, it is

not possible at the present time to conclude

whether all these enzymes, each catalyzing a

Pictet–Spengler type condensation, evolved from

the same ancestor. Although, the amino acid

sequence of norcoclaurine synthase an enzyme

which also catalyses a Pictet–Spengler reaction

(Samanani et al. 2004), is not apparently related

to STR1 and so differences in enzymatic mech-

anism cannot be described. It seems on the

basis of sequence that norcoclaurine synthase

belongs to a different enzyme family (the

pathogenesis-related (PR) 10 and Betv1 family),

but it is still possible so far that this synthase

also uses the same reaction mechanism as STR1.

If so, the different families of enzymes catalyzing

Pictet–Spengler reaction are examples of con-

vergent evolution. A clear-cut answer to this

question can only come from inspection of the

3D-structure of norcoclaurine synthase, which is,

not yet available.

Family members and evolutionary aspects

of STR1

There are a number of gene-related members in

the STR1 family, but up to now primary struc-

tures of only four STR1s are known; two from

Rauvolfia species and one each from the genus

Catharanthus and Ophiorrhiza. Sequence align-

ment studies reveal that all the major elements

such as the hydrophobic core of the b-propeller,

the hydrophobic binding pocket, the characteris-

tic disulfide bond and the catalytic Glu309 are

conserved throughout these family members. It is

therefore very likely that during evolutionary

divergence maintained their active site architec-

ture. Moreover, it seems very likely that nature

has used the STR1 scaffold for a great variety of

functions, because STR1-like genes are found in

plants and animals which are not able to perform

alkaloid biosynthesis. In Arabidopsis a whole

STR1-like gene family is known with 26 to 39%

sequence identity to STR1, but functions of these

genes have not been determined. Therefore de-

tailed structural and functional comparisons with

and within this gene-related family are impossible

at the present time.

The 6-bladed b-propeller is a well known pro-

tein fold, although not isolated from plants. Be-

cause of this fact it was interesting to compare the

STR1 structure with other b-propellers in order to

search for any evolutionary relationships. It is

noteworthy that STR1 has no significant sequence

identities or any functional similarities with other

6-bladed propellers. There are only very few en-

zymes known showing significant structural simi-

larities such as diisopropylfluorophosphatase

(DFPase, Scharff et al. 2001), serum paroxonase

(PON1, Harel et al. 2004), brain tumor NHL

Phytochem Rev (2007) 6:15–34 21

123

domain (NHL, Edwards et al. 2003) or the low

density lipoprotein receptor (LDLR, Jeon et al.

2001), but structure based alignments with the

STR1 structure indicated, only 11% to 16%

identity between them.

Inspection of the amino acid sequence in

LDLR identified a repetitive X-Tyr-Trp-Thr-Asp

(X is a hydrophobic amino acid) motif. In the

sequence of the remaining enzymes, five similar

motifs were found in the STR1 sequence which

might point to an evolutionary relationship of

these proteins. Although, two sequences in STR1,

X-Leu-Val-Ala-Glu and X-Trp-Val-Ser-Ser on b-

B4 and b-B5 seem to be not related to the con-

served X-Tyr-Trp-Thr-Asp repeat, closer inspec-

tion of these sequences would suggest a common

motif (X-X-X-#-§) for all of the b-propeller folds

discussed here (Ma et al. 2006). This motif is a

cluster of three hydrophobic amino acids (X-X-

X) followed by a small (#, Ser/Thr/Ala/Val/Gly)

amino acid and a hydrophilic residue (§, mostly

Asp or Glu).

This observation points to an evolutionary

relation of all these structurally similar proteins.

It is likely that STR1, LDLR, DFPase, NHL and

PON1 have evolved from one ancestral gene.

Duplication of such a b-sheet gene might lead to

the 6-bladed b-propeller structure containing the

X-Tyr-Trp-Thr-Asp repeat. During the evolution

single mutations might have shaped the binding

pocket and generated the different functions of

these proteins but the overall architecture of the

fold has been maintained. Structure-based

sequence alignment of STR1 and other 6-bladed

b-propellers from none-plant origin together with

the common motif X-X-X-#-§ described are here

displayed in Fig. 2e.

Based on the pronounced amino acid sequence

diversity and the different functions of ‘‘unre-

lated’’ propeller structures, it has been suggested

that these enzymes have evolved from distinct

ancestral b-sheet genes (Jawad and Paoli 2002).

The 3D-structure of STR1 and the structure-

based alignment between STR1 and the other

b-propeller genes mentioned here allow us to

instead propose an evolutionary relationship

between these apparently unrelated 6-bladed

b-propeller structures.

Strictosidine glucosidase (SG), crystallization and

X-ray analysis

The enzyme which follows STR1 in the pathway

is strictosidine glucosidase (SG) (Fig. 1). This

glucosidase ‘‘activates’’ the first intermediate,

strictosidine, by de-glucosylation. The resulting

aglycone is highly reactive and, cannot be directly

isolated. It is a precursor for all the pathways

leading to the entire family of monoterpenoid

indole alkaloids. Therefore the second enzyme of

the pathway also occupies an important role at

early stages of monoterpenoid indole alkaloid

biosynthesis of which ajmaline is one. Initially SG

has been detected and partly characterized from

cell suspension cultures of Catharanthus roseus by

Treimer and Zenk (1979) and was subsequently

described several times. The SG cDNA from

R. serpentina was isolated using the homology

cloning technique (Gerasimenko et al. 2002),

which was based on the SG cDNA sequence from

C. roseus reported by Geerlings et al. (2000). The

enzyme shows high substrate specificity and,

based on its primary structure, it belongs to the

family 1 of glycosyl hydrolases. Since a detailed

explanation of the enzyme reaction and the sub-

strate acceptance could not be found from se-

quence alignment studies we planned to

crystallize SG after overexpression in E. coli in

order to establish the 3D-structure of the enzyme

from R. serpentina. The cloning of the cDNA of

SG was based on primer development from the

SG sequence of C. roseus and raucaffricine glu-

cosidase (RG) of R. serpentina. The expression

vector PTYB1 resulted in low expression in

E. coli, and consequently the vector pQE-2 was

used in E. coli M15 cells. This strategy delivered

sufficient amounts of pure SG (up to 5 mg en-

zyme per liter bacterial culture) after His-tag

cleavage and MonoQ chromatography for crys-

tallization experiments. After testing most of the

commercially available crystallization kits it

turned out that formation of needles (Fig. 3a) was

easily achieved but the size and quality of these

crystals were not useful for X-ray analysis. Fur-

ther screening of crystallization conditions finally

gave good diffracting crystals (Fig. 3b) (Barleben

et al. 2005). Crystals of SG belonged to space

22 Phytochem Rev (2007) 6:15–34

123

group P4212. The structure of SG was determined

by using the molecular replacement technique

since SG was predicted to be a (b/a)8 barrel. This

so-called TIM-barrel is named after the structure

of triosephosphate isomerase, which was the en-

zyme which structure defining this fold (Henrissat

and Bairoch 1996; Gerlt and Raushel 2003). This

barrel is widely distributed in nature and the

Fig. 3 Strictosidine glucosidase (SG) (a) Needle crystalsof SG which were not useful for X-ray analysis. (b) Gooddiffracting crystal of SG resulting in resolution of 2.48 A.(c) The overall 3D-structure of Rauvolfia SG determined

from crystallized enzyme functionally expressed in E. coli.The structure represents a TIM-barrel fold in a gradientstarting from N-terminal to C-terminal in blue to redcolour

Phytochem Rev (2007) 6:15–34 23

123

enzymes with this fold catalyze many different

reactions and show only low sequence homology

(Henrissat and Bairoch 1996; Gerlt and Raushel

2003).

This fold is characterized by eight parallel

b-sheets located inside the enzyme. Connection

between the b-strands is reached by a-helices. The

overall topology is similar to a barrel. The loops

at the tops of the barrel determine the substrate

specificity. The glycoside hydrolase (GH) enzyme

super-family has been divided into four clans GH-

A, GH-D, GH-H and GH-K with a total of 24

different families. SG has been classified as

belonging to family 1 the GH-A clan. In this

family 16 enzymes have been structurally defined

of which only six are from plants, including the

two glucosidases from Rauvolfia, SG and RG. In

SG catalytic acid/base (Glu-416) is localized on

strand b-7 and the nucleophile Glu-207 in strand

b-4. Such an arrangement is typical for clan GH-

A glycoside hydrolases. The detail structural

analysis of SG will be reported in the near future.

Vinorine synthase (VS) sequence alignmentand site-directed mutations

The first enzyme generating the complete carbon

skeleton of ajmaline is named vinorine synthase

(VS). This enzyme is located in the middle of the

pathway (Fig. 1) and catalyzes an acetylCoA-

dependent ring closure of the product (16-epi-

vellosimine) of the preceding enzyme, polyneuri-

dine aldehyde esterase (PNAE). The product of

VS is the ajmalane-type alkaloid, vinorine, exhib-

iting the basic six-membered ring system of ajma-

line. VS has been previously detected in

R. serpentina cell culture. These cultures contain a

great number of acetylated alkaloids, such as vin-

orine, vomilenine and acetyl norajmaline. Acety-

lation in general should be dependent on activated

acetic acid and indeed, only in the presence of

acetylCoA are protein preparations from Rauvol-

fia cells able to convert 16-epi-vellosimine into

vinorine. VS has recently been highly enriched

(~1000-fold) by a five-step chromatographic pro-

cedure and its cDNA could be isolated from

R. serpentia · Rhazya stricta hybrid culture.

Functional expression in E. coli (Gerasimenko

et al. 2004; Bayer et al. 2004) was then possible.

Availability of the primary structure of VS allowed

the first sequence alignment studies. Figure 4 rep-

resents the alignment of VS with recently cloned

acetyltransferases of enzymes participating in

the biosynthesis of various highly valuable plant-

derived natural products such as acetylated

salutaridinol, an intermediate of morphine bio-

synthesis, vindoline which is important for the

formation of vinblastine and vincristine or acety-

lation of taxadienol on the biosynthetic way to the

cytotoxic diterpenoid alkaloid taxol. It includes

also other acyltransferases and highlighted the

conserved His-X-X-X-Asp sequence together with

Asp-Phe-Gly-Trp-Gly motif.

All these cDNA-derived proteins clearly

showed their sequence relationship by highly

conserved motifs such as Asp362-Phe-Gly-Trp-

Gly366 in VS. This motif is completely conserved

in all the above mentioned enzymes which are

involved in acetylCoA-dependent transforma-

tions during the above mentioned pathways. Also

the His160-X-X-X-Asp164 motif is 100% con-

served in these sequences in addition to the motif

Ser29-X-L/I-Asp32. In the latter case one excep-

tion exists which is for the enzyme deacetylvind-

oline acetyltransferase which has asparagine

instead of leucine or isoleucine in the motif at

N-terminus.

These sequence analyses indicated that VS

belongs to a small but constantly growing group

of enzymes, which have been called as the BAHD

enzyme super family (for a recent review see

D’Auria 2006). BAHD is the abbreviation for the

first four enzymes assigned to this family. These

enzymes were named Benzylalcohol acetyl-,

Anthocyanin-O-hydroxy-cinnamoyl-, anthrani-

late-N-Hydroxy-cinnamoyl/benzoyl- and Deace-

tylvindoline acetyl transferase. Our extensive

mutation experiments on VS have clearly identi-

fied His160 and Asp164 as the most important

amino acids for the catalytic process, because

their replacement by Ala completely knocked out

the vinorine synthase activity (Bayer et al. 2004).

Fig. 4 Structure based sequence alignment of the acetyl-CoA- dependent enzymes from plants belonging to theBAHD enzyme super family including putative acyltrans-ferases

c

24 Phytochem Rev (2007) 6:15–34

123

Phytochem Rev (2007) 6:15–34 25

123

26 Phytochem Rev (2007) 6:15–34

123

Moreover, Asp362 located in the consensus

sequence must also possess an important function

(see previous paragraph). Its replacement by Ala

significantly reduced the enzyme activity. The

same observation was made when the amino acids

Ser29 and Asp32, members of the newly desig-

nated consensus sequence at the N-terminus of

the enzyme, were exchanged by Ala (Bayer et al.

2004).

In a recently proposed classification of acyl-

transferases, all these enzymes were divided into

four evolutionary sequence clusters A–D (Hoff-

mann et al. 2003). In this classification system VS

would belong to the class C which includes

enzymes catalyzing acetylation of unrelated nat-

ural products. Class C is still very small but is

expected to increase since acetylation is a basic

reaction occurring frequently in biosynthesis. In

the Arabidopsis genome project already more

than 60 genes have been identified which also

might be members of the BAHD enzyme family

although they are still awaiting identification of

their metabolic functions.

The detailed knowledge recently accumulated

on catalytic domains, on novel motifs and on the

participation of single amino acids in the forma-

tion of vinorine will help to understand the acyl-

transfer reaction of the remaining enzymes of the

BAHD super-family. However, the mutation

experiments we carried out clearly suggested a

complicated reaction mechanism because we

identified at least seven amino acids reducing the

VS activity by varying degree. Unraveling the

catalytic process of acetyltransfer should now be a

major aim for enzymes of this particular family.

Crystallization of VS and X-ray analysis

Sequence comparison yields limited information

on reaction mechanism and because there was no

3D-structure known in this particular enzyme

family which could help us to understand the

mechanism, we developed crystallization condi-

tions for VS in order to analyze the molecular

structure of the transferase by X-ray crystallog-

raphy. The protein for the crystallization trials

was obtained after expression of the His-tagged

proteins in E. coli and purification by Ni-NTA

chromatography. Removal of the His-tag was

followed by ion exchange and size-exclusion

chromatography. Crystallization experiments by

hanging drop vapor-diffusion were performed

using, the commercially available Crystal Screen

and Crystal Screen 2 kits from Hampton Re-

search (USA). Only small multiple crystals, not

useful for X-ray measurements, were formed in

presence of ammonium sulfate and polyethylene

glycol (PEG) 400 at room temperature. Optimi-

zation trials varying precipitant concentrations,

type of PEG, enzyme concentration, buffer,

temperature and pH turned out to be essential for

diffraction quality crystal formation. The best

crystals with dimensions of about 0.3 · 0.15 ·0.1 mm were grown at typical conditions namely

at 32�C and at very low enzyme concentration

(2 mg ml–1) only (Ma et al. 2004b). It is interest-

ing to note, that in the Biological Macromolecule

Crystallization Database (Gilliland et al. 1994) no

example exists where a protein has been crystal-

lized at such low enzyme concentration combined

with such a high temperature. Crystallization of

VS also provided a nice example to the question

whether His6-tagged or the native proteins should

be used for crystallization trials, because both

were available in our case. In fact, it turned out

that the His6-tag enzyme precipitated or formed

needle clusters whatever conditions were used,

whereas the native synthase gave single crystals

that diffracted to a resolution of 2.8 A (Fig. 5a).

Dynamic light scattering experiments were per-

formed to explain this phenomenon. Results

indicated that VS exists as a monodisperse species

whereas the His6-tag VS tends to form aggregates

which do not crystallize.

Data for the flash-frozen VS crystals which

belongs to space group P212121, were measured,

however the 3D-structure determination was not

possible using molecular replacement. In order to

use the multiple-wavelength anomalous disper-

sion (MAD) approach to obtain experimental

phases, selenomethionyl VS (Se-Met VS) was

produced by adding selenomethionine to the cul-

ture broth (van Duyne et al. 1993). The Se-Met

substituted VS could be crystallized (Ma et al.

2005a) under similar conditions as for native VS

and X-ray measurements gave a complete set of

Fig. 4 continuedb

Phytochem Rev (2007) 6:15–34 27

123

data to 3.2 A at each of three wavelengths around

the selenium edge, allowing elucidation of the

3D-structure of the synthase using Auto-Rickshaw

pipeline (Panjikar et al. 2005) as described in (Ma

et al. 2005b) and refined to 2.8 A resolution.

Overall structure of VS

The atomic model of VS exhibits all amino acids

except residues 1–3 at the N-terminus and 5–6

residues in a loop located at the surface of the

enzyme (Ma et al. 2005b). In a total, this novel

protein structure exhibits 13 helices (a1–a13), 14b-

strands (b1–b14) and consists of two major do-

mains (A and B) with nearly identical size. Both

domains are connected by a large loop formed by

residues 201–213 (Fig. 5b). Six b sheets, b1, b2, b5,

b6, b7 and b12, represent domain A which is a

mixed six-stranded b sheet structure, covered on

both sides by seven helices (a1–a7). Domain A

also has a pair of b strands (b3 and b4) on the

enzyme surface. The second domain B, is a mixed

6-stranded b-sheet with b8–b11 and b13–b14. A

loop from domain B extends into domain A and

both domains have a very similar backbone fold,

but exhibit different topology.

A solvent channel running through the whole

VS molecule is formed by two loops, loop ‘‘a’’ and

loop ‘‘b’’. Loop ‘‘b’’ lies between strands b11 and

Fig. 5 Vinorine synthase (VS). (a) Single crystals of VSobtained with 2.0 M ammonium sulphate, 2% PEG 400and 100 mM Tris–HCl. (b) Overall structure of VS withCoA modelled into the active site. Helices and b-strandsincluding loops ‘‘a’’ and ‘‘b’’ are labelled. (c) Front view

down into the reaction channel of VS. Blue colourindicates the His160 residue necessary for the reaction.(d) Back view into the reaction channel with the bluecoloured His160 residue and each domain surface is shownin two different colours

28 Phytochem Rev (2007) 6:15–34

123

b13 of domain B but in addition includes b12 of

domain A. The second loop ‘‘b’’ is located between

b9 and b10. In loop ‘‘a’’ the motif Asp362-Phe-Gly-

Trp-Gly366 is situated. The Gly-Asn motif, also

completely conserved in the family, can be found in

loop b. The catalytic site motif His160-X-X-X-

Asp164 is located between both domains, so that

the catalytic His160 is in principal accessible from

both sides of the solvent channel (Fig. 5c–d).

The 3D-structure of VS together with the

mutational analysis yield detailed knowledge and

understanding of the catalytic mechanism, espe-

cially allowing differentiation between amino acid

residues important for catalytic and structural

reasons. The catalytic His160 of the His160-X-X-

X-Asp164 motif is located directly in the center of

the solvent channel. Therefore both reaction

partners, acetylCoA and 16-epi-vellosimine, may

approach the catalytic center independently from

each side of the channel. Previous kinetic data on

enzyme-catalyzed vinorine synthesis suggested

formation of a ternary complex between VS, sub-

strate and co-substrate by independent binding of

the ligands (Pfitzner et al. 1986). The structure of

VS is in excellent agreement with these biochem-

ical data. Asp164, the second crucial residue for

enzyme activity, points away from the active site

and is involved in a salt-bridge with Arg279.

Obviously, Asp164 has a structural function by

maintaining the geometry of the binding pocket.

Indeed this structural rather than catalytic signifi-

cance for Asp164 has been also discussed for other

acyltransferases (Buglino et al. 2004; Gibbs et al.

1990). The full conservation of the His-X-X-X-

Asp motif for all BAHD family members, they all

suggest that have a similar structural arrangement

of these residues and a similar reaction mecha-

nism. For a clear cut decision on their catalytic

functions and the structural properties of the

binding sites the 3D-structures of additional

BAHD members would be a prerequisite. The

second conserved motif of VS (Asp362-Phe-Gly-

Trp-Gly366) is unique to the BAHD super family

and was suggested to have functions in catalysis or

in binding of the co-substrate CoA (St-Pierre and

DeLuca 2000; Suzuki et al. 2003; Bayer et al.

2004). The 3D-structure presented here shows that

this motif is far from the active site pocket and is,

therefore not directly involved in catalysis. Instead

the motif is localized in a turn of a large loop

between b strands 11 and 12 in domain B. Most

probably this arrangement helps to keep the loop

in position which extends up to the pocket, thus

maintaining the shape of the enzyme’s catalytic

site and giving substrate specificity. In order to

attain more detailed and direct evidence on ligand

binding and atomic details of the reaction mech-

anism of VS, the crystal structures of substrate-

and product-bound complexes must be deter-

mined, work which has now been performed in our

laboratory.

Structural relationships of VS

The structure of VS can be classified in a general

way, as a member of CoA-dependent acyltrans-

ferases. A structural alignment in addition to a

sequence alignment offers the opportunity to find

relationships to other enzymes especially of those

of different origin and with different functions.

When the entire VS molecule is aligned using a

secondary structure matching servers (http://

www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver) the

most related structure found to be polyketide

synthase associated protein 5 (PDB code 1q9j)

from Mycobacterium tuberculosis, followed by

condensation domains of vibriobactine synthetase

(PDB code 115a), rat choline acetyltransferase

(PDB code 1q6x) and human carnitine acetyl-

transferase (PDB code 1nm8). When isolated A

and B domain of VS were used as a search model

the highest structural similarity was to chloram-

phenicol acetyltransferase (PDB code 1cla) and

dihydrolipoyl transacetylase (PDB code 1ead and

1eab) respectively. In fact, all these proteins are

CoA-dependent acyltransferases and the align-

ment results further confirm that VS is a member

of BAHD acyltransferase enzyme super family.

Details of the structural analysis of VS have been

recently described (Ma et al. 2005b).

Raucaffricine glucosidase (RG), crystallizationand X-ray analysis

Raucaffricine glucosidase (RG) is one of the en-

zymes of a side routes to the ajmaline biosyn-

thetic pathway. RG delivers in a one-step reaction

Phytochem Rev (2007) 6:15–34 29

123

the pathway intermediate vomilenine by de-glu-

cosylation of raucaffricine (Fig. 1). The concen-

tration of raucaffricine in Rauvolfia cell

suspension cultures exceeds that in the plant by a

factor of 67 and might accumulate to levels of up

to 1.6 g/l (Ruyter and Stockigt 1991).

RG was first detected and characterized two

decades ago (Schubel et al. 1986). Subsequently

RG has been purified from R. serpentina cell

suspension cultures with a 1200-fold enrichment.

The isolated enzyme suggests a precise value of a

molecular mass of 61 kDa and is not glycosylated.

After digestion with the endoproteinase LysC, six

peptide fragments were sequenced with a length

of 6–24 amino acids (Warzecha et al. 1999).

Based on these partial sequences the cDNA for

RG with an open reading frame of 1623 nucleo-

tides was isolated (Warzecha et al. 2000). The

corresponding protein consists of 540 amino acids

with a calculated molecular weight of 60931 Da in

agreement with molecular weight of the enzyme

isolated from the cell suspension. Comparison

with known glucosidase sequences shows, that

RG belongs to the family 1 of glycosyl hydrolases

(Henrissat 1991) and shares a sequence identity of

56% with SG.

For heterologous expression, RG cDNA was

cloned into the pSE280 vector (Warzecha et al.

2000). The recombinant RG shows high substrate

specificity with raucaffricine as the best substrate

(Km 1.3 mM, Vmax 0.5 nkat/lg protein). Only

some structurally related alkaloids like 1,2-di-

hydroraucaffricine and strictosidine are also de-

glucosylated. This observation is interesting since

RG is able to hydrolyse the substrate of strictos-

idine glucosidase (SG) where as SG is unable to

de-glucosylate raucaffricine, the substrate of RG

(Warzecha et al. 2000; Gerasimenko et al. 2002).

No hydrolysis for structurally different glucosides

such as loganin, secologanin, arbutin, vanillin-

b-D-glucoside or 4-nitrophenyl-b-D-glucoside has

been observed.

To obtain optimum expression RG cDNA was

cloned into the pQE-2 vector, which has been an

efficient vector in our work and expressed in the

E. coli strain M15 (Ruppert et al. 2006). For

easier isolation RG was expressed with an

N-terminal His6-tag and purified by using a

Ni-NTA column. With this method up to 5 mg

pure RG could be isolated per litre of bacterial

culture, and was directly suitable for systematic

crystallization experiments. Crystals of RG were

obtained by the hanging-drop vapour diffusion

technique at 20�C with 0.3 M ammonium sul-

phate 0.1 M sodium acetate buffer pH 4.6 and

11% PEG 4000 as precipitant (Ruppert et al.

2006). Crystals reach a maximum dimensions of

about 0.2 · 0.15 · 0.05 mm (Fig. 6a) belong to

space group I222 and diffract to 2.30 A (Fig. 6b)

with unit-cell parameters of a = 102.8 A,

b = 127.3 A and c = 215.8 A. The crystals were

treated with cryoprotectant, and were flash-

cooled to 100K prior to X-ray diffraction data

collection. Self-rotation analysis showed two

molecules per crystallographic asymmetric unit

although RG functions as a monomer in solution.

The preliminary X-ray analysis has been carried

out recently (Ruppert et al. 2006) and the detail

analysis of the enzyme is underway.

Summary, conclusions and future prospects

Phytochemical work in the previous years has

extensively broadened our knowledge on the

impressive structural complexity and diversity of

monoterpenoid indole alkaloids generated in

Rauvolfia cell and tissue cultures. With the iso-

lation and identification of more than 35 alkaloids

occurring in amounts in ranging from the lower

mg range up to 1.6 g per litre cell suspension. The

Rauvolfia systems represent one of the best

investigated biological systems of alkaloid for-

mation. This phytochemical characterization was

the prerequisite to reach the stage of identifying

and describing the alkaloidal metabolism of

Rauvolfia in detail, because the proteins involved

in catalyzing each step in the main route and in a

number of the side reactions of the ajmaline

biosynthetic pathway could be detected. From a

biosynthetic point of view a straightforward

metabolic route to the end product (e.g. ajmaline)

cannot in general, be expected. All examples

known so far show highly branched pathways,

resulting in a complicated metabolic network

which allows the metabolic diversity of secondary

products in nature. One possibility to steer a

pathway into the direction of a desired product,

30 Phytochem Rev (2007) 6:15–34

123

would be blocking specific side routes with a

specific inhibitor at the enzyme or cDNA stage

and generating in vivo the end product of the

main pathway, for instance ajmaline in the

Rauvolfia system. However, the specific genes or

enzymes must be known in order to utilize this

strategy.

The part of the Rauvolfia genome responsible

for alkaloid biosynthesis is now becoming avail-

able by use of a reverse genetic approach. The

cDNAs coding for soluble enzymes have been

isolated allowing heterologously overexpression

and functional characterization.

In most cases expression was achieved in

E. coli and judging from functional assays no

problems with incorrect protein folding could be

detected. Moreover, in spite of a few cases where

formation of inclusion bodies was observed, there

was always sufficient active enzyme produced for

the work summarized in this article. In fact, the

generation of a single enzyme of the ajmaline

pathway at a high purity (>98%) at mg-levels has

proven not to be problematic.

Having substantial amounts of these re-

combinant enzymes in hand one could think

about an enzyme directed biochemical synthesis

of intermediates of the pathway on preparative

scales, up to 100 mg or even higher. Compared

with classical product isolation from plants, a

step-wise synthesis with matrix bound enzymes

using regenerated coenzymes might be an alter-

native source of natural products in future. Such

an in vitro approach would however be exclu-

sively applicable for valuable products for which

natural sources are limited or those occurring

only in trace amounts in nature. If such an enzy-

matic process is developed, a serious restriction

could be the long term stability of the enzymes.

The development of stable, artificial enzymes

should be taken as a future challenge in many

research fields, including enzyme catalyzed syn-

thesis of natural products. The prerequisite of

such a strategy must be the elucidation of the

three dimensional structures followed by recog-

nition of the catalytic center of each catalyst to-

gether with the identification of any reaction

channel and an understanding of the reaction

mechanism. Understanding substrate recognition

and conversion at an atomic level could then lead

to a rational modulation of substrate specificity by

Fig. 6 Raucaffricine glucosidase (RG). (a) RG crystalfrom Rauvolfia serpentina and corresponding diffractionimage. (a) A close view of a single RG crystal obtained

after three days, with the dimensions of 0.2 · 0.15 ·0.05 mm and (b) image of X-ray diffraction pattern

Phytochem Rev (2007) 6:15–34 31

123

site-directed mutagenesis of the corresponding

cDNA. Enzymes in alkaloid biosynthesis are in

general highly substrate specific. One of our fu-

ture targets is to change this specificity in order to

obtain a desired substitution pattern at the indole

binding site of alkaloids. As discussed in this

chapter we have solved 3D-structures of several

of the major enzymes of ajmaline biosynthesis

since 2004, including the enzymes strictosidine

synthase and strictosidine glucosidase. Both en-

zymes are of an outstanding importance because

they function as the biosynthetic gate to the entire

monoterpenoid indole alkaloid family in higher

plants. The rational engineering and design of

both catalysts is now under investigation in our

laboratories.

Acknowledgements We are very thankful to Mrs. DorisRohr of our research group for cultivation of all plant cellsystems, to Prof. F. Lottspeich and his coworkers Mrs. I.Mathes and Mr. R. Mentele (Max-Planck-Institute ofBiochemistry, Martinsried, Germany) for their enthusiastichelp in sequencing pre-purified Rauvolfia enzymes and toProf. H. Michel, Dr. G. Fritzsch and Dr. J. Koepke (Max-Planck-Institute of Biophysics, Frankfurt/Main, Germany)for introducing us to structural biology research.

Our research was also supported by the DeutscheForschungsgemeinschaft (Bonn, Bad-Godesberg, Ger-many), Fonds der Chemischen Industrie (Frankfurt/Main,Germany) and the Bundesministerium fur Bildung undForschung (BMBF), Bonn, Germany). Support in theform of access to synchrotron facilities by the EuropeanCommunity (Research Infrastructure Action under theFP6 ‘‘Structuring the European Research Area Pro-gramme’’, contact number RII3/CT/2004/5060008) isalso acknowledged and we thank Dr. Paul Tucker(EMBL Hamburg, Germany) for critically reading themanuscript.

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