Structural Biology in Plant Natural Produc Biosynthesis. Architectur of Enzymes From Monoterpenoid...

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REVIEW www.rsc.org/npr | Natural Product Reports

Structural biology in plant natural product biosynthesisarchitecture ofenzymes from monoterpenoid indole and tropane alkaloid biosynthesis

Joachim Stockigt*a,b and Santosh Panjikarc

Received (in Cambridge, UK) 2nd August 2007

First published as an Advance Article on the web 24th October 2007DOI: 10.1039/b711935f

Covering: 1997 to 2007

Several cDNAs of enzymes catalyzing biosynthetic pathways of plant-derived alkaloids have recently

been heterologously expressed, and the production of appropriate enzymes from ajmaline and tropane

alkaloid biosynthesis in bacteria allows their crystallization. This review describes the architecture of

these enzymes with and without their ligands.

1 Introduction

2 Strictosidine synthase (STR1)

2.1 Biological PictetSpengler reaction and the role of

STR1 for the entire indole alkaloid family

2.2 Crystallization and structure determination of STR1,

and the Auto-Rickshaw software pipeline

2.3 The 6-bladed 4-stranded b-propeller fold of STR1the

first example from the plant kingdom

2.4 Structures of STR1 in complex with its substrates and

product

2.4 Substrate specificity of STR1 and mutagenesis studies

2.6 Structure-based alignments and evolution of STR1

3 Strictosidine glucosidase (SG) and its role

3.1 Optimization of crystallization of SG

3.2 Overall 3D-structure of SG

3.3 Site-directed mutagenesis and SG

Glu207Gln-strictosidine complex

3.4 Recognition of the aglycone and glycone part of

strictosidine

3.5 Substrate binding site of SG compared to those of

other plant glucosidases

3.6 Prospects for the use of SG for alkaloid synthesis

4 Polyneuridine aldehyde esterase (PNAE)

4.1 PNAEone of the most substrate-specific esterases?

4.2 Inhibition, primary structure and site-directed

mutagenesis of PNAE

4.3 The long route to PNAE crystals

4.4 Homology modelling and structure of PNAE

5 Vinorine synthase (VS)

5.1 The role of VS in generating the ajmalan structure

5.2 Inhibitors and mutants of VS

5.3 Unusual crystallization conditions and instability of

VS crystals

5.4 Structure elucidation of VS

aCollege of Pharmaceutical Sciences, Zijingang Campus, Zhejiang Univer-sity, 310058, Hangzhou, ChinabInstitute of Pharmacy, Johannes Gutenberg University Mainz, StaudingerWeg 5, D-55099, Mainz, GermanycEuropean Molecular Biology Laboratory Hamburg, Outstation DeutschesElektronen-Synchrotron, Notkestrasse 85, D-22603, Hamburg, Germany

In memory of Professor Pierre Potier.

5.5 Important amino acids and mechanistic aspects

5.6 Significance of the vinorine synthase structure for the

BAHD enzyme family

6 Raucaffricine glucosidase (RG), a side route of the

ajmaline pathway

6.1 Crystallization attempts and the 3D-structure of RG

7 Structure-based redesign of enzymes and applications

in chemo-enzymatic approaches

8 Other structural examples from the alkaloid field

9 Conclusions and future aspects of structural biology in

the alkaloid field

10 Acknowledgements

11 References

1 Introduction

Both their highly complex chemical structures and their pro-

nounced pharmacological activities have made research on al-

kaloids, including elucidation of their biosynthetic pathways, very

attractive for many decades. Major progress in the research into

the biosynthesis of plant monoterpenoid indole alkaloids has been

made by investigations of single enzymes, partial biosynthetic

routes and entire pathways.13 This has been sporadically reviewed

during the last 10 years together with the genetics of alkaloid

biosynthesis.4,5

There are, however, only very few examples from the alkaloid

field,which deliver at present a coherentknowledge of biosynthetic

routes at the enzyme level. These include:

(a) The well investigated enzymatic steps connecting the Aspi-

dosperma alkaloids tabersonine and vindoline, reactions occurring

at the periphery of the basic Aspidosperma alkaloid skeleton.4,610

(b) The biosynthesis of camptothecin, our knowledge of which,

despite some recent enzymatic work on the early steps,11 comes

largely from Hutchinsons group, a long time ago. 12,13

(c) The whole sequence between tryptamine plus secologanin

and the heteroyohimbines ajmalicine and some of its isomers

(Corynanthe alkaloids) was successfully investigated at the enzy-

matic level a few decades ago.1417

(d) Efficient alkaloid-producing cell suspension cultures of

the Apocynaceae plant Catharanthus roseus, established at the

1382 | Nat. Prod. Rep., 2007, 24, 13821400 This journal is The Royal Society of Chemistry 2007

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Joachim Stockigt received a PhD in organic chemistry from M unster University (Germany) with Professor Burchard Franck. He has

worked with Professor Meinhart H. Zenk at the Faculty of Biology (Bochum University, Germany) and the Faculty of Pharmacy (Munich

University, Germany) and is currently Full Professor at the Institute of Pharmacy (Mainz University, Germany; College of Pharmaceutical

Sciences, Zhejiang University, Hangzhou, China). His research interests include natural products biosynthesis (phytochemistry, enzymology,

molecular and structural biology).

Joachim Stockigt Santosh Panjikar

Santosh Panjikar earned a PhD in Biotechnology from Friedrich-Schiller University

(Jena, Germany) and held a postdoctoral appointment at the EMBL Hamburg

Outstation with Dr Paul A. Tucker. He is currently Staff Scientist at the EMBL

Outstation. His research interests focus on structure-based drug design, method

developments in structural biology and synchrotron instrumentation.

beginning of the 1970s in Zenks laboratories, became the key for

studies of this plant.18,19

(e) Cell suspension cultures of the Indian medicinal plant

Rauvolfia serpentina Benth. ex Kurz from the same lab were also

the major prerequisite for the isolation of some uncommon indole

alkaloids20 and elucidation of the pathway for the formation of

the antiarrhythmic ajmaline and structurally related alkaloids of

the sarpagan- and ajmalan-type both by identification of many

single enzymes2123 and in part by in vivo NMR.24,25 The number

of enzyme-catalyzed reactions proven in that Rauvolfia cell system

amounts to more than 15, representing at the moment one of the

most detailed investigations in alkaloid biosynthesis. The pathway

between tryptamine plus secologanin and ajmaline is illustrated in

Scheme 1.

Several of the cDNAs coding for enzymes of the above-

mentioned ajmaline route have been detected, isolated and func-

tionallyexpressed, mostly by usingthe reverse genetic approach,

applying polymerase chain reaction (PCR) and heterologous

systems, especially Escherichia coli. For instance, most of the

soluble Rauvolfia enzymes are now functionally expressed and

characterized in detail,23 especially with regard to their substrate

specificity and theirmajor kinetic data and general properties,such

as temperature and pH optimum, molecular size, and isoelectric

points. For the first time, cloning has also provided amounts of

enzyme, in almost pure form, on the 2050 mg scale by fusion-

protein techniques. This is the amount necessary to develop

crystallization conditions of these proteins and to elucidate their

three-dimensional structure by X-ray analysis.

The following article summarizes in detail the achievements

in the elucidation of the architecture of five major enzymes

of the ajmaline biosynthetic pathway in Rauvolfia,26 namely

strictosidine synthase (STR1), strictosidine-O-b-D-glucosidase

(SG), polyneuridine aldehyde esterase (PNAE), vinorine synthase

(VS) and raucaffricine glucosidase (RG), together with two

tropinone reductases (TR-I and TR-II) from tropane alkaloid

biosynthesis.27,28 Other than a short general overview on 3D-

analysis of Rauvolfia enzymes,26 this is the first comprehensive

and detailed review on structural biology in the alkaloid field,

providing a more direct insight into the mechanisms of alkaloid

biosynthesis.

2 Strictosidine synthase (STR1)

2.1 Biological PictetSpengler reaction and the role of STR1 for

the entire indole alkaloid family

Strictosidine synthase (STR1, EC 4.3.3.2), which catalyzes the

stereoselective PictetSpengler reaction of tryptamine and secolo-

ganin to form 3a(S)-strictosidine, has been described many times

since its original detection.29,30,3134 It was the first enzyme from

alkaloid biosynthesis whose cDNA was functionally, heterolo-

gously expressed.3537 Later, the synthases from several plants other

than Rauvolfia,35 such as Catharanthus38,39 and Ophiorhiza,40 were

functionally expressed. The primary importance of STR1 is not

only its precursorrole forthe biosynthetic pathway of ajmaline, but

also because it initiates, in fact, all pathways leading to the entire

monoterpene indole alkaloid family. Some prominent members

are depicted in Fig. 1. Although STR1 is the crucial enzyme for all

the 2000 monoterpenoid indole alkaloids, little information about

its reaction mechanism and the critical amino acids for enzymeactivity was known.

2.2 Crystallization and structure determination of STR1, and the

Auto-Rickshaw software pipeline

Expression and crystallization of STR1 was straightforward and

typical of our crystallization of other Rauvolfia proteins, using

pQE-2-plasmid and M15 E. coli cell line. The hanging-drop

vapour-diffusion technique was used routinely and was the most

efficient method in our hands. STR1 crystals were in space

group R3 with a hexagonal unit cell containing two molecules

of the enzyme. A set of selenium-labelled STR1s (4SeMet- and

This journal is The Royal Society of Chemistry 2007 Nat. Prod. Rep., 2007, 24, 13821400 | 1383

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Scheme 1 Enzyme-catalysed biosynthesis of ajmaline in cell suspension culture of the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz.

Abbreviations are: STR1 = strictosidine synthase, SG = strictosidine glucosidase, SBE = sarpagan bridge enzyme, PNAE = polyneuridine aldehyde

esterase, VS = vinorine synthase, VH = vinorine hydroxylase, CPR = cytochrome, P450 reductase, VR = vomilenine reductase, DHVR =

dihydrovomilenine reductase, AAE= acetylajmalan esterase, NAMT=norajmalan methyltransferase, RG= raucaffricine glucosidine,VGT=vomilenine

glucosyltransferase. Strictosidine aglycone has not been isolated. Enzymes shown in bold have been heterologously expressed, those marked with asterisks

have been crystallised and their 3D-structures determined recently in our laboratories. They are described in this review.

Fig. 1 Formation of strictosidine and its biosynthetic role as central

precursor of various monoterpenoid indole alkaloids.

6SeMet-STR1)needed to be preparedin order to solve theenzyme

structure, because no protein structures with significant sequence

homology to strictosidine synthase were available.41,42 The STR1

structure was finally solved using the multiple wavelength anoma-

lous dispersion (MAD) approach together with software package

Auto-Rickshaw,43 and refined to 2.4 A.

Structures of all enzymes from the ajmaline biosynthetic

pathway discussed in this article were determined using Auto-

Rickshaw. In fact, early evaluation of the software pipeline was

performed on several multiple wavelength diffraction (MAD)

datasets from crystals of various proteins, including MAD data

of vinorine synthase (VS) and strictosidine synthase (STR1). At

a later stage, native structures or ligand complexes of the various

enzymes from the pathway were determined routinely using Auto-

Rickshaw. Here we describe briefly the software pipeline which

provides a means of rapid structure solution of proteins.

Crystal structure determination both by isomorphous replace-

ment and by anomalous scattering techniques is a multi-step

process in which each step, from substructure determination to

model building and validation, requires certain decisions to be

made. These decisions comprise the choice of the crystallographic

computer programs that are most suitable to perform the specific

tasks andthe optimal input parameters for each of these programs.

The interpretability of the map depends to a large extent on the

success of the preceding steps and is generally limited by the

resolution of the data and the quality of the phase information.

Traditionally, each of the steps described was carried out by an ex-

perienced crystallographer, whose skill manifested itself in finding


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the optimum, or at least a successful, path towards the completion

of the structure determination. The automated crystal structure

determination platform combines a number of macromolecular

crystallographic software packages with several decision-making

steps. The entire process in the pipeline is fully automatic. Each

step of the structure solution is governed by the decision making

module within the platform, which attempts to mimic the decisions

of an experienced crystallographer. The role of computer-coded

decision-makers is to choose the appropriate crystallographic

computer programs and the required input parameters at each

step of the structure determination. Various phasing protocols are

encoded in the system. These are single anomalous diffraction

(SAD), single isomorphous replacement with anomalous scatter-

ing (SIRAS), multiple wavelength diffraction (MAD), standard

molecular replacement (MR), phased MR and combination of

MR and SAD. A large number of possible structure solution

paths for each phasing protocolare encoded in thesystem, andthe

optimal path is selected by the decision-makers as the structure

solution evolves.

Once the input parameters are given (number of amino acids,

heavy atoms, molecules per asymmetric unit, probable space group

and phasing protocol) and X-ray data have been input to Auto-

Rickshaw, no further user intervention is required.It proceedsstep

by step through the structure solution using the decision makers.

The Auto-Rickshaw server (http://www.embl-hamburg.de/Auto-

Rickshaw) is available to EMBL-Hamburg beamline users, and

the server will be made more widely accessible in the immediate

future.

2.3 The 6-bladed 4-stranded b-propeller fold of STR1the first

example from the plant kingdom

The overall structure of STR1 belongs to the six-bladed four-

stranded b-propeller fold, where the blades are radially located

around a pseudo-six-fold symmetry axis. Each blade consists of

twisted four-stranded b-sheets. Although this particular fold has

been detected in different organisms several times, this is the first

example from the plant kingdom. However, the other enzymes

with the six-bladed fold have a completely different function. 44,45

The active site of STR1 is near to the symmetry axis, as shown by

the structure of the enzymetryptamine complex (Fig. 2).46 There

are only three helices in the STR1 structure, of which two are

connected by an SS bridge, which is essential for both the overall

structure and the shape of the catalytic centre.

2.4 Structures of STR1 in complex with its substrates and

product

Tryptamine is located deep in the binding pocket, sandwiched

between two hydrophobic residues (Tyr151 and Phe226) holding

the molecule in the correct orientation for the PictetSpengler con-

densation. In addition, Glu309, which is important for the STR1

activity as proven by mutagenesis experiments, is coordinated with

the amino group of tryptamine. There are (primarily) hydrophobic

amino acids lining the binding pocket, and both positively charged

and hydrophobic residues are located at the entrance to the binding

site of STR1.

The complexstructureof STR1bound withsecologaninexhibits

the monoterpene in the same binding pocket (Fig. 3a). The

Fig. 2 Strictosidine synthase (STR1) from Rauvolfia in complex with its

substrate tryptamine (N and C mark the N- and C-termini of the protein;

the arrow points to the SS bridge).

hydrophilic glucose unit of secologanin points out of the catalytic

pocket towards the solvent. This complex is illustrated in Fig. 3b,

which is rotated by 84 along the x-axis to show better the

accessibility of the hydrophilic part of the monoterpene to the

solvent. The aldehyde group of secologanin points towards Glu309

and is in close proximity to the amino group of tryptamine

(as observed in the previous structure), ready for the primary

condensation reaction.

The third complex was prepared by soaking crystals of STR1 in

a solution containing strictosidine, and the structure illustrates

how the product of the STR1 reaction is accommodated in

the catalytic centre (Fig. 4). The location of the strictosidine is

very similar to that found for both substrates (see above) but

is not totally identical, showing that the way the ligands are

accommodated is to some extent flexible before and after the

reaction. The electron density gives additional information about

how the product is shielded from the residues of the active centre.

Such shielding might have important implications for the substrate

specificity of STR1 and should help in structure-based rational

design of substrate acceptance.

2.4 Substrate specificity of STR1 and mutagenesis studies

As far as the substrate specificity of STR1 is concerned, several

studies with both STRs from Catharanthus and Rauvolfia have

been reported earlier.31,4651 We have analyzed the substrate ac-

ceptance of STR1 again and focused especially on explanations

of why particular tryptamine derivatives were not accepted by

STR1 based on the available structural information. Table 1

highlights the comparison of substrate specificity of STR1,

especially for tryptamines substituted differently at position 5.

These tryptamines are not accepted in the case of 5-methyl- and

5-methoxy-derivatives by the wild-type enzyme.52 The complex

structure demonstrates that the Val208 side-chain is shielding the


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Fig. 3 (a) Strictosidine synthase (STR1) in complex with its substrate

secologanin. (b) Fig. 3a rotated by 84 around the x-axis.

5-position, preventing binding of 5-substituted indole bases. Re-

placementof thisparticular Val by the smaller Ala clearly broadens

the substrate acceptance for 5-substituted tryptamines, delivering

novel strictosidine analogues.52 Further structure-derived muta-

tions basedon thisstructureare expectedto have significantimpact

on future enzymatic synthesis of new strictosidines structurallymodified at the tryptamine or secologanin parts.

Moreover, if the enzyme strictosidine glucosidase (SG), which

follows STR1 in the biosynthesis of all the monoterpene alkaloids,

can be modified in a similar way to that described for the synthase,

future chemo-enzymatic approaches might be developed for the

generation of novel alkaloid libraries with biologically important

compounds.

2.6 Structure-based alignments and evolution of STR1

STR1 has no functional homologies to other six-bladed b-

propeller folds. Also, a structurally based sequence alignment of

Fig. 4 3D-structure of STR1 in complex with its product strictosidine.

Table 1 Substrate acceptance of STR1 for differently substitutedtryptamines at positions 5 and 6 (n.d. = not detectable, data taken inpart from ref. 52)

Substrates Enzymes Km/mM (kcat/Km)/mM1 s1

Tryptamine Wild-type 0.072 147.92Val208Ala 0.219 246.99

5-Methyltryptamine Wild-type n.d. Val208Ala 0.281 23.35

5-Methoxytryptamine Wild-type n.d.

Val208Ala 3.592 22.186-Methyltryptamine Wild-type 0.393 5.90Val208Ala 0.762 14.37

6-Methoxytryptamine Wild-type 0.962 5.53Val208Ala 0.307 54.27

5-Fluorotryptamine Wild-type 0.259 144.63Val208Ala 1.302 16.31

6-Fluorotryptamine Wild-type 0.136 171.84Val208Ala 0.356 38.29

5-Hydroxytryptamine Wild-type 2.255 249.29Val208Ala 0.844 21.47

these folds showed less than 16% sequence identity with most b-

propellers such as diisopropylfluorophosphatase (DFPase)45 from

Loligo vulgaris, brain tumour NHL domain,53 serum paroxonase44

and low-density lipoprotein receptor YWTD domain. Analysis of

the STR1 fold compared to the just-mentioned folds indicates that

these structurally related proteins are also related from the point

of view of their evolution.46 Most probably they all evolved from

an ancestral b-sheet gene. The ancestral structure might have been

modified by e.g. deletions or insertions during evolution, leading to

a differently shaped reaction pocket in order to adopt the different

functions of these propellers. These propellers keep a common

sequence homologywhich maintainsthe overall structuralfeatures

of the protein fold. It is typical of these six-bladed b-propellers

that they are diverse in sequence and function and that they are of

different phylogenetic origin.


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A similar reaction to that of STR1 found in the plant Alangium

lamarckii Thw. (Alangiaceae) is catalyzed by deacetylipecoside

synthase (DIS). This synthase catalyzes the condensation between

dopamine and secologanin, forming (1R)-deacetylipecoside which

has the opposite configuration to strictosidine (at C-3).54 DIS

and STR1 are similar with respect to their reaction type (Pictet

Spengler condensation), their pH optimum, temperature optimum

and their molecular size. Both enzymes exhibit high substrate

specificity but they come from different plant species and families,

and exhibit different substrate specificity and stereo-selectivity. It

cannot be excluded, however, that both synthases evolved from the

same ancestor but during evolution developed different substrate

specificities leading to different alkaloid types. Cloning of DIS and

comparison of the 3D-structures of both enzymes could help to

support this hypothesis.

3 Strictosidine glucosidase (SG) and its role

Similarly to STR1, strictosidine glucosidase (SG, EC 3.2.1.105)

has been described several times from Catharanthus and from

Rauvolfia.

5557

Here we refer mainly to the Rauvolfia enzymebecause it was the second protein from the ajmaline biosynthetic

pathway to be crystallized. The general and major importance of

SG lies in its function to chemically activate strictosidine by

deglucosylation. The aglycone thus generated is highly unstable

and reactive. It enters all the pathways to the various structural

types illustrated in Fig. 1, but the exact molecular stage at

which it initiates all these pathways has not been clarified so far.

Although glycosides are involved in many metabolic processes

such as regulation of plant hormone activity and biosynthesis58,59

and lignification,60 one of the main functions of glycosidases,

especially in higher plants, is believed to be defence-related.61 For

example, the generation by glycosidases of toxic products such

as cyanide or isothiocyanates from the appropriate glycosidesmakes theirecological significance as defensive agents immediately

understandable.62 SG has also been discussed as being defence-

related since its product(s) were shown to have antibiotic activity.56

However, in view of the important role that the aglycone of

strictosidine plays as the biogenetic precursor of all of the 2000 or

so monoterpenoid indole alkaloids, a synthesis-related function

of SG seems to be more important than its defence-related role.

Reasons for the pronounced substrate specificity and details

of the reaction mechanism of this enzyme remained mostly

unknownuntil recently. Therefore, an efficientover-expression and

purification system for strictosidine glucosidase in Escherichia coli

was developed, followed by crystallization and preliminary X-ray

analysis of the enzyme.

3.1 Optimization of crystallization of SG

When our routine expression system (E. coli, M15 [pREP4])

and purification method (fusion protein with N-terminal His 6-

tag), followed by removing the tag with dipeptidyl aminopeptidase

(DAPase) and final enzyme purification by MonoQ ion exchange

chromatography was used, about 10 mg of pure SG were obtained

from a 5 litre culture of E. coli. Such an amount of enzyme

is acceptable for a large screening of crystallization conditions.

More than 10 commercially available crystallization kits were

tried. Many conditions resulted in formation of rather flat- or

needle-shaped crystals (Fig. 5a), which were not useful for X-

ray experiments. However, in a large trial using a Cartesian

syn QUADTM Microsys crystallization robot with 600 nl sitting

drops, two conditions were found to produce rectangular crystal

plates. These conditions gave, after extensive optimization with

the hanging drop method, improved crystals for X-ray mea-

surements with a final resolution of 2.48 A (Fig. 5b).63

Fig. 5 (a) Needle-type crystals of SG not suitable for X-ray analysis.

(b) A rectangular prism of SG (images are from ref. 26).

3.2 Overall 3D-structure of SG

From initial alignment studies SG unequivocally belongs to the

glycosyl hydrolase (GH) family 1, which is grouped together with

16 other families in clan GH-A, representing the biggest of the13 clans of glycosidases. There are only seven three-dimensional

structures of glucosidases from family 1 known from eukaryotic

sources, and six of these are of plant origin. The expression,

purification, crystallization and preliminary X-ray analysis of SG

has recently been reported.63 In Fig. 6 the overall fold of SG

Fig. 6 Tim barrel overall fold of SG (2.48 A).


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is shown, representing the well known (b/a)8 barrel fold. The

structure is built from 13 a-helices and 13 b-strands; the core of

the structure consists of 8 parallel b-strands that form a b-barrel.

This barrel is surrounded by eight helices and hosts the catalytic

binding site of SG for the substrate strictosidine.

3.3 Site-directed mutagenesis and SG Glu207Gln-strictosidine

complex

Complete conservation of amino acid residues Glu207, Glu416

and His161 is indicated by sequence alignment of SG with

glucosidases of various origins. Glu207 is the proton donor that

assists nucleophilic attack of Glu416 at the anomeric carbon C-1.64

Hydrolysis of the glucoside bond is catalyzed in concert by both

glutamic acids.65 For SG no enzyme activity was obtained after

mutations Glu207Gln, Glu207Asp, Glu416Gln or Glu416Asp.66

This clearly points to the crucial role of both glutamates for the

deglucosylation of strictosidine. In the reaction catalyzed by SG,

His161 also plays an important role. When it is replaced by Asn

or Leu, enzyme activity is decreased to


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Fig. 8 Comparison of the substrate complex of SG (a) with those of two

other b-glucosidases, from Zea mays (b) and from Sorghum bicolor (c),67,68

displaying the different conformation of tryptophan (W376) in SG. This

illustration was partly taken from ref. 66 (hydrophobic residues in grey,

hydrophilic in green, acidic residues (Glu, Asp) in red, positively charged

residues in blue).

3.6 Prospects for the use of SG for alkaloid synthesis

A future demand for natural products, in this case of complex

plantalkaloids with high structuraldiversity, will probably depend

on detailed biosynthetic knowledge, on rational molecular engi-

neering of the enzymes, their synthetic application and metabolic

engineering of the medicinal plants.70,71 Because of the special and

multiple role of the aglycone of strictosidine, the enzyme SG may

serve as an attractive candidate for such directed biosynthesis

approaches. Such approaches only make sense if the substrate

specificity of SG can be modified. Because the three-dimensional

structure of strictosidine synthase has become available, initial

steps were successful to engineer and to change the substrate

acceptance of STR1.52 Generation of large alkaloid libraries by

structure-based enzyme re-designof STR1 and SG in combination

with biomimetic approaches may therefore become possible in the

near future.47,51,52

4 Polyneuridine aldehyde esterase (PNAE)

4.1 PNAEone of the most substrate-specific esterases?

Polyneuridine aldehyde (PNA) is the first alkaloid in the pathway

to ajmaline, exhibiting a sarpagan structure.72 The aldehyde

is biosynthetically generated by the so-called sarpagan-bridge

enzyme (SBE),73 whose mechanism has not been elucidated so far.

Theesterase PNAE (EC3.1.1.78) converts PNA after ester hydrol-

ysis and decarboxylation into 16-epi-vellosimine.71,74 The substrate

specificity of this enzyme has been investigated several times at

various stages of enzyme purity,72,74,75 and was finally determined

with the overexpressed homogenous His6-tag PNAE.Froma series

of 13 structurally different esters only the natural substrate (PNA)

was hydrolyzed (Fig. 9).7678 The esters included ten methyl esters,

of whicheight were monoterpenoid indole alkaloids and two more

simple methyl esters, and additionally three acetate esters. The

results demonstrated an exceptionally high substrate specificity of

the over-expressed enzyme and indicated that PNAE might be one

of the most substrate-specific esterases.

4.2 Inhibition, primary structure and site-directed mutagenesis of

PNAE

Inhibition studies with selective serine or cysteine inhibitors

and unselective histidine/cysteine inhibitors provided insight into

reactive residues of the enzyme (Table 2). They showed complete

inhibition with diethyl pyrocarbonate, indicating the importance

of histidine for enzyme activity, and partial inhibition was ob-

served with serine/cysteine inhibitors. However, these experiments

did not allow PNAE to be defined as a serine or cysteine

hydrolase. Site-directed mutagenesis was applied to get more

detailed information on particular amino acids that seemed likely

to be involved in the catalytic process of PNAE from a rigorous

analysis of the primary structure of the enzyme. Comparison of

Table 2 Inhibition of PNAE by serine, cysteine, and histidine inhibitors

Inhibitor Type of Inhibition Concentration Incubation time/min Relative inhibition (%)

AEBSF Selective Ser 4.0 mM 60 0E-64 Selective Cys 25 lM 60 0TPCK Ser-Cys 200 lM 60 12PMSF Ser-Cys 1.0 mM 60 20DEPC Unselective His 1.2 mM 60 100Hg2+ Unselective Cys 200 lM 60 100


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Fig. 9 Structurally different esters as putative substrates of PNAE. Only the natural substrate, polyneuridine aldehyde, displayed any activity. 78

the overall amino acid sequence of PNAE showed relatively high

identity (up to 50%) to some putative lyases e.g. from Arabidopsis

thaliana.79 A 4043%identitywas foundfor two well characterized

hydroxynitrile lyases (Hnls), one from Hevea brasiliensis (HbHnl)

and one from Manihot esculenta (MeHnl)8087 with known 3D-

structure, demonstrating a relatively close relationship of PNAE

to Hnls (Scheme 2). The above enzymes belong to the a/b-fold

hydrolases, indicating that PNAE is a newly-detected member of

that enzyme superfamily. The most important features of thatfamily are a catalytic triad formed by a nucleophilic residue, an

acidic amino acid and a histidine, which appear in the same order

in PNAE (Ser87, Asp216, His244), and two additional strongly

related and conserved motifs. Indeed, mutations in PNAE showed

the presence of the catalytic triad, since replacement of each of the

three residues by alanine resulted in completely inactive mutants,76

classifying the enzyme as a novel a/b-fold hydrolase. Structural

analysis of PNAE (see Section 4.4) supported that classification.

4.3 The long route to PNAE crystals

The key to the crystallization of an enzyme is its purity. Crystal-

lization also depends on the availability of homogenous, ideallymonodisperse solutions of protein molecules with a uniform,

rigid three-dimensional fold. Protein crystallization can be divided

into two steps: coarse screening to identify initial crystallization

conditions and then optimization of these conditions to produce

Scheme 2 Alignment of the primary sequence of PNAE with hydroxynitrile lyases, showing the close relationship of the three enzymes, the conserved

catalytic triad (marked with asterisks) and two typical motifs (black on red) around His17 and Ser87.


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single, diffraction-quality crystals. Currently, there are no system-

atic methods to ensure that ordered three-dimensional crystals will

be obtained.88 Sitting-drop and hanging-drop methods of vapour

diffusion are the most commonly used techniques. Microbatch

crystallization under oil is mainly used when these crystallization

methods have failed.89 A recent development in protein crystal-

lization has been the use of microfluidic systems for crystallizing

proteins using the free-interface diffusion method on the nanolitre

scale, but this has one major drawback in that it is often difficult

to translate hits into higher volume solutions in order to grow

crystals for diffraction experiments.

Crystal optimization aims to turn poor quality crystals into

diffraction-quality crystals that can be used for structure deter-

mination. There are a variety of methods that can be used to

improve crystal quality, including crystal seeding. The nucleation

event in protein crystallization is a poorly understood process.

In many crystallization experiments, it is not possible to reach

sufficiently highlevels of saturation fornucleus formation. In many

cases, the introduction of a crystal or crystal seed stock at lower

levels of saturation can facilitate nucleation and crystal growth.

Micro-, macro-, heterogeneous and in situ seedings represent

different seeding techniques. High-throughput crystallization and

visualization platforms have been widely established and are com-

monly used by high-throughput structuralgenomics initiatives.9093

Crystallization experiments can be monitored using an imaging

robot and the images can either be analyzed manually or using

automatic crystal recognition systems. Such a kind of system

has been installed at EMBL Hamburg, and the high-throughput

crystallization facility has been open to the general scientific

community.92 The facility covers every step in the crystallization

process from the preparation of crystallization cocktails for initial

or customized screens to the setting up of hanging-drop vapour-

diffusion experiments and their automatic imaging.

Obtaining useful PNAE crystals was our most tedious example

of plant protein crystallization because it took in total four years

to obtain good X-ray-diffracting crystals, although the entire time

period was not invested in this particular enzyme. The two major

obstacles were: (a) precipitation of PNAE in crystallization solu-

tion and (b) the complete lack of crystal (nucleus) formation when

nearly all of the commercially available crystallization kits were

applied. Most of the typical crystallization procedures mentioned

above were used. In a total about 6000 crystallization conditions

were tried, with very few conditions resulting in crystal formation.

Only 2-dimensional crystals were obtained, which were useless

for X-ray analysis. These conditions then were optimized in time-

consuming trials by hand-pipetting of 48 ll hanging drops.

From about 1000 drops, 10 crystals were finally detected, from

which the best gave a resolution of 2.0 A and allowed immediate

structure elucidation of the enzyme. Meanwhile, the success rate

in crystallizing this particular enzyme increased and probably will

also become routine soon.

4.4 Homology modelling and structure of PNAE

The structure of PNAE was modelled76,77 based on the 3D-

structure of hydroxynitrile lyase (EC 4.2.1.39) from Hevea

brasiliensis, which was available at 1.9 A resolution (see Protein

databank 1YAS) and which has an amino acid sequence identity

to PNAE of 43%. Homology modelling gave a range of PNAE

models with the software MODELLER (version 4). 94 The model

with the best overall steric properties was chosen for comparison

with the 3D-structure of PNAE generated from X-ray data.

The 3D-structure of His6-PNAE (resolution of 2.1 A) was

elucidated by the molecular replacement method with the

software Auto-Rickshaw.43 The model of the salicylic acid

binding protein 2 (SABP2, pdb code: 1XKL) from Nicotiana

tabacum95 with identity of the amino acid sequence to PNAE

of 54% was successfully used as search model for molecular

replacement. Sufficient electron density was not observed for the

His6-tag or for the first eight amino acids from the N-terminal

end of the enzyme because of high flexibility. The fold of PNAE

belongs to the a/b-hydrolase family, which is one of the biggest

and fastest growing enzyme families with about 4250 members.96,97

Based on the 3D-structure, PNAE indeed represents a novel

member of that family. The enzyme consists of eight b-strands

and eight a-helices (Fig. 10).

Fig. 10 Overall structure of PNAE highlighting the cap-region, which

is shown by the arrow.

The two anti-parallel strands and the three helices (see sec-

ondary structure above, the straight line in Fig. 10) form a domain

which is named the cap region. The connection of all these

structural elements is illustrated by the 2D-topological diagram

(Fig. 11). The three amino acids (Ser87, Asp216 and His244)

that form the catalytic triad and are essential for enzyme activity,

as shown by mutation experiments, are included in Fig. 11.77

The enzyme structure illustrates that these residues are located

directly in the reaction channel. Ser87 is positioned in the so-

called nucleophilic elbow which is located between the b3 sheet

and a3 helix. The narrow reaction channel connects the binding

site with the surface of the enzyme, and the channel is flanked by

predominantly hydrophobic residues of the cap-domain. This

domain consists of a total of 71 amino acids from Asp116 to

Phe187. In accord with the non-polar structure of the substrate

PNA, about 50% of the cap consists of hydrophobic residues.

Most of the a/b-hydrolase enzymes exhibit this domain, which is

involved in substrate recognition and interfacial activation.98 The

cap-region has, in contrast to the core-region of these enzymes,


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Fig. 11 2D-topological diagram of PNAE illustrating the connection of

the structural elements (cap domain at bottom).

a spectacular variability in topology, leading to the surprising

variability in functions and to the substrate selectivity, which is

especially pronounced for PNAE.

Comparison of the overall structure of modelled PNAE withthe X-ray structure gave a root mean square deviation (rmsd)

value of 0.916 A. For the cap region it was 1.045 A, and the

core region exhibited a rmsd of 0.812 A. The core region,

the conserved region in the a/b-hydrolase family, shows the lower

rmsd, whereas the flexible and variable cap domain differs more

between the two models (rmsd of 1.045 A).

Comparison of the catalytic amino acids Ser87, Asp216 and

His244 shows only slight differences between the three X-ray

models of PNAE, HbHnl and the salicylic acid binding protein.

The entrance to the channel is represented in Fig. 12 and

shows clear differences between the modelled and the X-ray-

derived structure of PNAE, indicating the necessity of the X-ray

analysis. There is a future need to generate appropriate substrate

enzyme complexes to understand more precisely the extraordinary

substrate specificity of the enzyme and especially to get to know

details about the amino acids involved in the recognition of the

substrate PNA.

5 Vinorine synthase (VS)

5.1 The role of VS in generating the ajmalan structure

On the pathway to the ajmalan basic skeleton, vinorine synthase

(VS, EC 2.3.1.160) plays a central role by finalizing the synthesis

of the six-ring ajmalan system.The enzymeconverts thesarpagan-

type alkaloid epivellosimine, which is the reaction product of

the preceding enzyme PNAE (see Section 4), to the ajmalan-

type in a coenzyme A-dependent reaction. VS has been identified

from R. serpentina cell suspensions and its properties and were

preliminarily described a long time ago.99 Later, the cDNA of theenzyme was isolated and could successfully be expressed in E. coli.

Again, the reverse genetic approach was the successful strategy

to get the full length cDNA clone:100,101 after purification of VS

from the plant cells, it was identified by SDS gel electrophoresis

and partial sequencing. The primary structure of the encoded

protein and sequence alignmentstudies demonstrated that it was a

novel enzyme. The pQE-2 vector and E. coliM-15 cell line served

for good overexpression with mg amounts of enzyme per litre

Fig. 12 Geometry of the entrance of the reaction channel (A) derived from X-ray analysis and (B) from modelling of PNAE; and zoomed regions (C)

and (D), respectively (images provided by Dr M. Hill).


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of bacterial suspension. The soluble enzyme was purified, as for

the previous proteins (Sections 24), by Ni-NTA chromatography,

always an important step in getting homogenous enzyme prepara-

tions forbiochemical characterization as wellas for crystallization.

The functional identity of VS was proven by showing formation of

the product of the reaction (the methoxylated indolenine alkaloid

vinorine) by electron impact mass spectrometry.101

Sequence alignment studies supported classification of VS

within the so-called BAHD enzyme superfamily.102 BAHD is

the abbreviated name from the first four enzymes of this family

isolated from plant species. Primarily this classification was

based on two highly conserved motifs in that family, 362Asp-Phe-

Gly-Trp-Gly366 and 160His-xxx-Asp164, respectively. As deduced

from X-ray structures of chloramphenicol acetyltransferase and

dihydrolipoamide acetyltransferase, these motifs participate in

the catalytic reaction of acetyl transfer.103,104 A catalytic triad

(Lys-His-Asp) was discussed for the activity of arylamine N-

acetyltransferase.105,106 The primary structure of VS contains

several of these residues, suggesting an important function of these

particular three amino acids.101

5.2 Inhibitors and mutants of VS

After expression of the VS cDNA in E. coli and its purification

to near homogeneity, inhibition studies gave the first clue as to

the catalytically active amino acids.101 Inhibitors acting on Ser,

His and Cys such as chloromethyl ketones, the unselective His-

directed diethyl pyrocarbonate, Hg2+-ion as a SH-group modifier,

the Ser- and Cys-selective benzenesulfonylfluroide (AEBSF) and

the agmatine derivative E-64 were all tested. All these compounds

inhibited the VS activity.101 The results clearly indicated that Ser,

His and Cys are essential for the VS-catalyzed reaction. Together

with results of sequence alignment studies, a series of mutation

experiments was performed101

and gave some detailed informationon the putative catalytic residues (see Table 3). Five conserved Ser

residues were replaced by Ala and all the derived mutant enzymes

still showed catalytic activity. This made involvement of a Ser-

His-Asp triad in the binding site unlikely. Also, when Cys89 and

Cys149 were mutated to Ala, the VS activity was not completely

knocked out, making a Cys-His-Asp triad also unlikely. But

replacement of His160 and Asp164 by Ala resulted in inactive

Table 3 Recombinant His6-tagged VS mutants and their relative activity(n.d.= not detectable, data are from ref. 101)

Enzyme Relative activity (%)

Wild-type 100S68A 100C89A 100S413A 100D360A 100S16A 71N293A 68D362A 35S29A 25S243A 17D32A 14C149A 10H160A n.d.D164A n.d.

enzyme mutants, which provided the first clear evidence for the

functional and maybe exclusive role of the BAHD-typical domain

His-xxx-Asp in this particular enzyme family. The second motif,362Asp-Phe-Gly-Trp-Gly366(DFGWG), is completely conserved in

the BAHD enzymes. Mutation of the first Asp reduced the VS

activity by about one-third, a result matching with mutation

of another BAHD enzyme involved in anthocyanin glucoside

malonylation.107 The 3D X-ray structure of VS discussed below

will explain the significance of this motif. Based on sequence

alignments, acyltransferases are divided into four evolutionary

sequence clusters.108 Adopting this classification, VS falls into

cluster C enzymes catalyzing esterification of hydroxyl groups of

metabolically unrelated secondary metabolites.

From the above results only two probable catalytically active

amino acids were identified for VS; His160 and Asp164. Their

function was proven by the 3D-structure of VS (discussed in

Section 5.4), after finding appropriate conditions of crystallization

of the recombinant enzyme in long crystallization trials.

5.3 Unusual crystallization conditions and instability of VScrystals

Beginners luck is definitely a factor in finding conditions for

crystallizing a protein the first time. This is partly because

beginners are more willing to try new conditions and will often

do nave things to the sample, thus finding novel conditions for

crystal growth. This is also because no one can predict the proper

conditions for crystallizing a new protein.109

In this light, VS represents a good example of successfully using

rather uncommon crystallization conditions. Similarly to the other

enzymes described in this article, on the one hand the purification

procedure of VS after over-expression in E. coli was crucial for

obtaining the first crystals, but on the other hand, removal of the

His6-tag by DAPase was the second requirement to succeed. Each

experiment to crystallize the His-tagged enzyme resulted in pre-

cipitation or in undetectable nucleation of the enzyme. After Ni-

NTA chromatography, two additional chromatographic steps (ion

exchange on MonoQ followed by Sephacryl S-100) were essential

andgave highly pure VS as judgedby SDSgel chromatography and

Coomassie staining. Also in this case the hanging drop method

was used and initially small, clustered crystals were observed

on applying Crystal Screen and Crystal Screen2 kits from

Hampton Research110,111 at 22 C and using ammonium sulfate as

the precipitant in the presence of polyethylene glycol (PEG) 400.

Optimum crystallization conditions were found after systematic

changes of precipitant concentrations, types of PEG, buffers, pH,

temperature and enzyme concentrations. VS crystals obtained

only at the relatively high temperature of 32 C and low protein

concentration of 2 mg ml1, proved to be the best ones for X-ray

measurements. These conditions are exceptional, because such a

combination of temperature and enzyme concentration was not

found in the Biological Macromolecule Crystallization Database

(BMCD),112 and was just based on trying unusual conditions.

The well diffracting VS crystals proved to be very sensitive to

temperature changes. In contrast to all other crystals mentioned

in this article, it was not possible to transport VS crystals for

synchrotron measurements without careful freezing procedures,

and such procedures were always essential.


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5.4 Structure elucidation of VS

Up to the time when this review was written, there were no

crystal structures known in the BAHD enzyme family which

could be helpful for 3D-structure elucidation of VS. By inhibition

of methionine biosynthesis,113 the recombinant E. coli produced

(in the presence of selenomethionine) SeMet-VS, which was

purified and crystallized similarly to the wild-type VS. The multi-

wavelength anomalous diffraction (MAD) approach togetherwith the diffraction data of the wild-type VS (2.6 A resolution)

allowed for the first time determination of the VS structure, for

which the selenium sites were taken as marker residues to place

the amino acid side chains into the appropriate electron densities.

After several rounds of refinement, the final three-dimensional

structure of VS was obtained.114116 The structure contains 14 b-

strands (b1b14) and 13 a-helices (a1a13) and consists of two

domains, A and B, of about the same size. The two domains are

connected by a long loop. Both have a similar backbone fold, but

their topology is different. Between the domains a solvent channel

is formed, running through the entire VS molecule (Fig. 13).

The His-xxx-Asp motif is located at the interface of the two

domains. His160 is situated directly in the centre of the solventchannel and is accessible from both ends of the channel. Such

an arrangement allows both reaction partners, 16-epi-vellosimine

and acetyl-CoA, to approach the active site. Kinetic data obtained

with a partially purified VS preparation from Rauvolfia cells had

earlier suggested a ternary complex between both ligands and the

enzyme with independent binding of the ligands.99 The mechanism

Fig. 13 Image of VS illustrating the two-domain structure (A and B), the

reaction channel and the catalytic His in the binding pocket.

of the reaction is illustrated in Scheme 3. The 3D-structure of VS

strongly supports this proposal.

5.5 Important amino acids and mechanistic aspects

The mutagenesis studies indicated the indispensability of His160

for VS activity, and the 3D-structure of the enzyme confirms

the functional importance of this residue. It is this amino acid

which is located directly in the centre of the solvent channel(Fig. 14). This is in agreement with the above-mentioned kinetic

data, that the two substrates may approach the binding pocket

from different directions (from which direction still needs to be

analyzed). Based on His160, the acetyl-transfer reaction would

occur as illustrated in Scheme 3. The amino acid acts as a base,

taking off the proton of the hydroxy group of 17-deacetylvinorine.

The 17-oxygen will then attack the carbonyl carbon of acetyl-

CoA, resulting in its acetylation and release of CoA. The reaction

probably proceeds without formation of an acetylated enzyme

intermediate. The second conserved residue (Asp164) is also

located directly at the binding site and belongs to the essential

His-xxx-Asp motif. Asp164 has no catalytic function. Its side

chain points away from His160, not allowing hydrogen-bondformation between the residues. Such an arrangement excludes

an amino acid dyad in the catalytic process as found for e.g.

human carnitine acetyltransferase.117 Asp164 forms a salt bridge

to Arg279, which is also a conserved residue in the BAHD family.

This interaction of thetwo residues appears to be of structural, not

of catalytic, importance. Exchange of Arg279 against Ala results

in loss of enzyme activity. Most probably it is the geometry of

the binding pocket which is significantly changed when the salt

bridge is interrupted. It must be demonstrated in future whether

the importance of the His-xxx-Asp motif in other enzymes of

the BAHD family is based on the same reasons as shown for

VS. The DFGWG motif completely conserved in that family is

not localized in the binding pocket of the enzyme. In contrast, itis far away from the region of catalysis, indicating its structural

importance. It is not involved in substrate binding and might

maintain the conformation of the enzyme structure. Mutation

of the Asp residue to Ala therefore caused decrease of VS activity

(Table 4). Such mutation also resulted in complete loss of reactivity

of another BAHD enzyme participating in anthocyanin glucoside

malonylation.107 A more detailed insight into thebindingpocket of

VS requires more structural information. In particular, the crystal

structure of vinorine synthase with substrate or product bound

should provide much deeper understanding of the catalytic process

in this enzyme family.

Scheme 3 Proposed reaction mechanism of VS; scheme from ref. 116.


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Fig. 14 Surface representation, reaction channel and the localization of

the catalytic residue His160 (in red) in VS (images kindly provided byDr M. Hill).

Very recently, structural and mutational studies on anthocyanin

malonyltransferase were reported.118 This enzyme is also a BAHD

member and now represents the second example where the 3D-

structure has been solved. The complex of this malonyltransferase

with its ligand malonyl-CoA together with a series of site-directed

mutations allowed the acyl-acceptor binding site to be identified.

These results together with those on VS are very helpful for the

understanding in more detail of the diversity of the acyl-acceptor

specificity within the BAHD family.118

5.6 Significance of the vinorine synthase structure for the BAHDenzyme family

The BAHD enzymes belong to a constantly growing family with

an increasing number of functionally expressed members. At the

DNA level it is suggested that in two organisms alone 180 genes

occur that might code for BAHD acyltransferases; in Oryza sativa

(rice) 119 genes of this family have been identified, but none have

been investigated for their biochemical function. The Arabidopsis

thaliana genome delivered 64 genes of the family, but very few

have been functionally described so far. The (Z)-3-hexen-1-ol

O-acetyltransferase (producing the acetylated hexenol)119 or

another anthocyanidin 5-O-glucoside-O-malonyltransferase120 are

recent examples. Rosmarinic acid synthase is one of the most

recent members of the BAHD family detected by reverse

genetics.121 Prediction of the reactions catalyzed or the substrates

accepted by the enzymes, based on sequence alignments, is

difficult.122 But mutation experiments, such as those described

above, combined with sequence comparison might help to elu-

cidate the functionalities of other members of the BAHD family.

The structure of VS could also be helpful in solving other

structures by molecular replacement approaches, or at least could

provide opportunitiesfor homology modelling. Thisis particularly

important for those family members for which the cDNAs have

functionally been cloned and which take part in the biosynthe-

sis of important plant natural products. Prominent examples

are salutaridinol acetyltransferase in morphine biosynthesis,123

deacetylvindoline acetyltransferase124 on the way to vinblastine,

and the acyltransferases acting on the skeleton of taxol,125 just to

mention BAHD members involved in alkaloid biosynthesis.

6 Raucaffricine glucosidase (RG), a side route of the

ajmaline pathway

As a part of a metabolic network, the ajmaline biosynthetic

pathway has at several intermediate stages side routes, of which

the formation of raucaffricine is one of the most important.

Raucaffricine is the glucoside of the intermediate vomilenine. This

glucoalkaloid has previously been detected as the main alkaloid

of Rauvolfia cell cultures, exceeding significantly the amounts

in differentiated Rauvolfia cells.126 The enzyme, raucaffricine

glucosidase (RG, EC 3.2.1.125), converts the glucoside back to

vomilenine. It has been identified, characterized and its cDNA has

been cloned in E. coli.127,128 RG represents the second glucoside-

hydrolyzing enzyme in Rauvolfia alkaloid biosynthesis, the first

being SG. The primary structure and the substrate specificity of

the two glucosidases are different; RG accepts strictosidine, thesubstrate of SG, but SG does not hydrolyze raucaffricine.127,128 To

compare the two glucosidases, it was important to determine the

3D-structure of RG in order to evaluate substrate recognition and

better understand the deglucosylation process. Over-expression

yielded enough pure RG enzyme for crystallization experiments

when the routine strategy, applied for theother Rauvolfia enzymes,

was followed.

6.1 Crystallization attempts and the 3D-structure of RG

His6-tagRG was crystallized by the hanging-drop vapour diffusion

technique using similar conditions as for strictosidine glucosidase.

Crystals reached maximum dimensions of about 0.2 0.15

0.05 mm. The crystals belong to space group I222 and diffract to

2.30 A.129

The structure of RG was determined by the molecular replace-

ment method (unpublished data). The structure of Rauvolfia SG

served as a search model, because RG shares a sequence identity

with SG of 56%. RG belongs to the family 1 of the glycosyl

hydrolases. Crystal packing showed two enzyme molecules for

each crystallographic asymmetric unit, but the enzyme is active as

the monomer.127

The refined model of RG consists of 13 a-helices and 13 b-

strands. RG adopts the expected topology of a single (b/a)8 barrel

fold (TIM barrel) (Fig. 15). The barrel hosts a binding site for the


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natural substrate raucaffricine. The groove leading to the catalytic

centre is formed mainly by irregular loops between the secondary

structures on top of the enzyme. Similarly to SG, RG contains the

catalytic residues E186 and E420. The most striking difference is

at the catalytic centre of the RG and SG. An identical position of

Trp392 but a different orientation of the tryptophan in the two

glucosidases helps the protein to recognize its substrate.

Fig. 15 The (b/a)8 barrel fold of RG, illustrating the binding pocket and

the catalytic acids E186 and E420.

7 Structure-based redesign of enzymes and

applications in chemo-enzymatic approaches

The enzymes described in this review are of relatively high

substrate specificity. This is a great disadvantage in terms of using

them in the future as biocatalysts e.g. for the enzymatic synthesis

of structurally novel alkaloids. For instance, the best substrate for

STR1 is tryptamine, and benzene-ring-substituted tryptamines

generally react at less than 10% of the rate for tryptamine. The

STR1 mutant Val208Ala, however, converts 5-methyltryptamine

and 5-methoxytryptamine into the corresponding novel substi-

tuted strictosidines, indicating the importance of knowing the 3D-

structure of STR1 for redesigning its enzyme activity.51,52 Based

on such information, a more systematic mutation approach will in

the future show whether a further expanded substitution pattern

at the indole moiety can be achieved.

A biomimetic approach which uses strictosidine (or derivatives),

strictosidine glucosidase and an excess of primary amines (with ob-

viously anyresiduesR followed by reduction) (Scheme 4), results

in formation of novel N-analogous heteroyohimbine alkaloids.47,52

After producing STR1 mutants (allowing synthesis of a range of

novel strictosidines) and optimizing the second enzyme strictosi-

dine glucosidase by mutation, such a chemo-enzymatic approach

may be an excellent tool for thegenerationof new alkaloid libraries

containing thousands of alkaloids. Knowledge of biosynthesis

combined with structural biology might be an excellent tool in fu-

ture to develop such combinatorial enzyme-mediated approaches.

8 Other structural examples from the alkaloid field

Despite the fact that alkaloids have been an excellent source

of biologically active compounds, e.g. anti-infectious drugs,130

only limited efforts have been focussed on understanding the

mode of action of alkaloids and their interactions with host

proteins or enzymes from other organisms at the molecular level.

These include crystallographic studies of tropinone reductase,

phospholipase, transcriptional repressor, acetylcholine esterase,

calmodulin and tyrosine kinase and their complexes with various

alkaloids, listed in Table 4.

Below the only other structural examples from alkaloid biosyn-

thesis, the tropinone reductases, are discussed in some detail.

Tropinone reductase (TR) comprises a branching point in the

biosynthetic pathway of tropane alkaloids which includes such

medicinally important compounds as hyoscyamine, scopolamine

and cocaine (Scheme 5). All the tropane-alkaloid-producing plants

species so farexamined havetwo TR activities andtheir amino acid

sequence is known from Datura stramonium,131,132 Hyoscyamus

niger,133 Atropa belladonna134,135 and Solanum tuberosum.136 In

these species, there are two types of TR, TR-I and TR-II. These

Scheme 4 A biomimetic approach to indole alkaloid diversity based on rational enzyme design (STR1, strictosidine synthase; SG, strictosidine

glucosidase; X, various substituents).


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Table 4 3D-structures of enzymealkaloid complexes from the PDB data bank

PDBcode Protein Complex with alkaloid

2AE2 Tropinone reductase-II Pseudotropine142

1IPF Tropinone reductase-II Tropinone143

1ZR8 Phospholipase Ajmaline144

1JUM Transcriptional repressor Berberine145

1VOT Acetylcholine-esterase Huperzine A146

1ZGB Acetylcholine-esterase (R)-Tacrine-C10H20-hupyridone

147

1DX6 Acetylcholine-esterase Galanthamine148

1H22 Acetylcholine-esterase (S)-Hupyridone-C10H20-(S)-hupyridone149

1XA5 Calmodulin A bis-indole alkaloid150

Scheme 5 Biosynthetic connection of tropane alkaloids through the intermediate tropinone (ODC, ornithine decarboxylase; PMT, putrescine

methyltransferase; TR-I, tropinone reductase I; TR-II, tropinone reductase II).

TRs share 64% sequence identity and belong to the short-chain

dehydrogenase/reductase (SDR) family. TRs catalyze NADPH-

dependent reductions of the 3-carbonyl group of their common

substrate, tropinone, to hydroxy groups with different diastere-

omeric configurations: TR-I (EC 1.1.1.206) produces tropine

(3a-hydroxytropane), and TR-II (EC 1.1.1.236) produces pseu-

dotropine (W-tropine, 3b-hydroxytropane). These enzymes have

different Km values for tropinone and its analogues buthavesimilar

Km values for NADPH, and both catalyze transfer of the pro-S

hydrogen atom of NADPH to tropinone.137

This indicates that the two TR enzymes have different binding

sites for tropinone but have similar ones for NADPH, and that

their different stereospecificities result from the different binding

modes of tropinone. Both TRs from Datura stramonium have been

cloned and expressed in E. coli, and purified and crystallized

using hanging drop vapour diffusion techniques with sodium

citrate and 2-methyl-2,4-pentenediol as buffer and precipitant

respectively.138,139 The structure of each TR was solved using the

isomorphous replacement method.140 The two structures are al-

mostindistinguishable from each other in bothsubunitfolding and

their association into dimers. Both TR subunits consist of a core

domainthat includes most of thepolypeptide and a small lobe that

protrudes fromthe core. In thecentre of thecoredomain is a seven-

stranded parallel b-sheet, flanked on each side by three a-helices,

which constitutes the Rossmann-fold topology. This core structure

is highly conserved among the SDR family members, despite

relatively low residue identity between these enzymes (30%).141

The structure of TR-I was also determined in the presence of

NADP+. The cofactor was found to be located at the bottom of

the cleft between the core domain and the small lobe. The carbox-

amide group of the nicotinamide ring is anchored by the main-

chain nitrogen and oxygen atoms of Ile204 and the side-chain


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oxygen of Thr206. This tight binding of the carboxamide group

to the protein directs the B-face of the nicotinamide ring toward

the void of the cleft, consistent with the observed specificity for

the pro-Shydride transfer of both TRs.137

TR-II catalyzes the one step chemical reaction via the following

states:

(1) TR-II (E) + NADPH (S1) + tropinone (S2)

(2) ES1S2

(3) EP1P2

(4) TR-II + NADP+(P1)+ W-tropinone (P2),

wherethe statesare: (1)E, thefreeenzyme; (2)ES1S2, theenzyme

with the substrates [NADPH (S1) and tropinone (S2)] bound prior

to reaction initiation; (3) EP1P2, the enzyme with the products

[NADP+ (P1) and W-tropine (P2)] bound just after the reaction

is completed; and (4) the free enzyme again after releasing the

products.

The crystallographic structures of TR-II have been determined

in an unliganded form,140 as the complex with NADPH,141 as the

complex with NADPH and tropine,141 and as the complex with

NADP+ and W-tropine.142 These structures provide a great deal

of insight into each state of the reaction in the biosynthesis of

tropane alkaloids (Fig. 16).

Fig. 16 (a) Complex structure of tropinone reductase II (TR-II) with

tropinone in black and NADPH in blue+grey. (b) Complex structure of

tropinone reductase I (TR-I) with NADPH illustrated in blue+grey.

9 Conclusions and future aspects of structural

biology in the alkaloid field

Elucidation of the three-dimensional structures of several of the

Rauvolfia enzymes has extensively broadened our knowledge on

details of the enzymatic biosynthesis of monoterpenoid indole

alkaloids, particularly of the pathway to the antiarrhythmic

ajmaline. For five major enzymes, the fold-families, their overall

structures and substrate binding sites are now known, including

identification of amino acids of catalytic and structural impor-

tance. This will facilitate elucidation of the corresponding enzyme

mechanisms. For enzymes of more general significance, such as

strictosidine synthase, crystal structures of substrates and product

complexes have been analyzed; this allows understanding of ligand

recognition and permitted the first successful rational enzyme

engineering for the synthesis of novel alkaloids. Structure-guided

combinatorial approaches with redesigned enzyme mutants to-

gether with biomimetic strategies will help to generate large

alkaloid libraries with hopefully important and novel biological

activities.52

10 Acknowledgements

We thank Mrs Yang Liuqing for kind help in preparing the

diagrams for this article. The described research was continuously

supported by the Deutsche Forschungsgemeinschaft (Bonn, Bad-

Godesberg, Germany), the Fonds der Chemischen Industrie

(Frankfurt/Main, Germany) in cooperation with the Bundesmin-

isterium fur Bildung und Forschung (BMBF, Bonn, Germany), the

DLR Office Bonn of BMBF, and the Deutscher Akademischer

Austauschdienst (DAAD), Bonn, Germany. The work was also

supported by European Community Access to Research Infras-

tructure Action of the Improving Human Potential Programme

to the European Molecular Biology Laboratory (EMBL) at Ham-

burg Outstation (contract No. HPRI-CT-1999-100017) and the

FP6 Programme (No. RII3/CT/2004/5060008), and the Berliner

Elektronenspeicherring-Gesellschaft fur Synchrotronstrahlung

(Berlin, Germany). We also acknowledge staff members at the

DORIS storage ring (DESY, Hamburg, Germany), Prof. Lottspe-

ichs team at the Max-Planck-Institute of Biochemistry (MPI,

Martinsried, Germany) for protein sequencing, Prof. H. Michels

group at MPI (Frankfurt/Main, Germany) and Prof. T. Kutchan

(St Louis, USA) for introduction into structural and molecular

biology techniques, respectively, and the ESRF beamline (Greno-

ble, France) for technical help. Present co-workers (in alphabetical

order: Dr Leif Barleben, Dr Marco Hill, Petra Kercmar, Elke

Loris, Kerstin Oelrich, and Dr Martin Ruppert) and former co-

workers of various nationalities are verymuch appreciated fortheir

enthusiastic performing of chemical,enzymatic and crystallization

experiments. Both Dr E. Mattern-Dogru and Dr XueyanMa orig-

inally initiated structural biology research in our group. We thank

Dr F. Leeper (Cambridge, UK) and Dr P. Tucker (EMBL Ham-

burg, Germany) for advice and help in correcting the manuscript.

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