Arbutin synthase, a novel member of the NRD1β glycosyltransferase family, is a unique multifunctional enzyme converting various natural products and xenobiotics

Article abstract of DOI:10.1016/S0968-0896(02)00029-9

Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics. We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6xHis tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50°C and showed two different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates comparable to hydroquinone were not achieved. In contrast to this broad acceptor substrate specificity, only pyrimidine nucleotide activated glucose was tolerated as a donor substrate. Nucleotide and amino acid sequence analysis revealed AS to be a new member of the NRD1β family of glycosyl transferases and placed the enzyme into the group of plant secondary product GTs. Arbutin synthase is therefore the first example of a broad spectrum multifunctional glucosyltransferase.

Full text of DOI:10.1016/S0968-0896(02)00029-9

Bioorganic & Medicinal Chemistry 10 (2002) 1731–1741
Arbutin Synthase, a Novel Member of the NRD1ꢀ
Glycosyltransferase Family, is a Unique Multifunctional Enzyme
Converting Various Natural Products and Xenobioticsy
Tobias Hefner,a Joachim Arend,a Heribert Warzecha,b
Karsten Siemsc and Joachim Stockigta,*
aJohannes Gutenberg-University Mainz, Institute of Pharmacy, Department of Pharmaceutical Biology,
Staudinger Weg 5, D-55099 Mainz, Germany
bBoyce Thompson Institute for Plant Research, Cornell University, Tower Road, 14850 Ithaca, NY, USA
cAnalytiCon Discovery, Hermannswerder Haus 17, D-14473 Potsdam, Germany
Received 19 November 2001; accepted 14 January 2002
Dedicated to Professor Leopold Horner on his 90th birthday.
Abstract—Plant glucosyltransferases (GTs) play a crucial role in natural product biosynthesis and metabolization of xenobiotics.
We expressed the arbutin synthase (AS) cDNA from Rauvolfia serpentina cell suspension cultures in Escherichia coli with a 6xHis
tag and purified the active enzyme to homogeneity. The recombinant enzyme had a temperature optimum of 50 ꢀC and showed two
different pH optima (4.5 and 6.8 or 7.5, depending on the buffer). Out of 74 natural and synthetic phenols and two cinnamyl
alcohols tested as substrates for the AS, 45 were accepted, covering a broad range of structural features. Converting rates com-
parable to hydroquinone were not achieved. In contrast to this broad acceptor substrate specificity, only pyrimidine nucleotide
activated glucose was tolerated as a donor substrate. Nucleotide and amino acid sequence analysis revealed AS to be a new member
of the NRD1b family of glycosyl transferases and placed the enzyme into the group of plant secondary product GTs. Arbutin
synthase is therefore the first example of a broad spectrum multifunctional glucosyltransferase. # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
soluble aglycones into water soluble glucoconjugates and
by enabling storage of the formed compounds within
the vacuole. Moreover, once de-toxified and compart-
mentalized these products can subsequently be hydro-
lyzed by specific enzymes. Upon cell destruction,
glucosidases break down these stable conjugates yield-
ing the toxic aglycones which might act as deterrents
against herbivores. Prominent examples of such a plant
defense mechanism are glucosinolates and cyanogenic
glucosides which are converted into highly reactive iso-
thiocyanates and the toxic hydrogen cyanide.1,2 An
even larger group of structurally diverse secondary meta-
bolites consists of phenolic glucosides whose aglycones
may serve as efficient enzyme inhibitors after oxidation.3
For natural products, the conjugation of one or more
sugar residues — glycosylation — constitutes an essen-
tial component of functional diversity. Glucosylation,
the transfer of activated glucose to an aglycone sub-
strate, is the predominant modification in plants, com-
pared to microorganisms or animals. Since this
enzymatic conjugation is in most cases the last step in
the biosynthesis of plant natural products it plays an
important role in stabilizing the product or in altering
its physiological activity. Plant systems typically retain
secondary metabolites intracellularly, therefore their
accumulation might interfere with basic physiological
functions, especially if the compounds are toxic or pre-
cipitate due to low solubility. Glucosylation helps cir-
cumvent these detrimental side effects by converting barely
The biosynthetic formation of all these glucosides is
catalyzed by a large enzyme family, named glycosyl-
transferases (GTs). Although a variety of GTs of plant
origin have been identified of which several have been
expressed heterologously, their characterization is still
very preliminary, especially regarding their substrate
specificities and active domains.4,5
*Corresponding author. Tel.: +49-6131-392-5751; fax: +49-6131-
392-3752; e-mail: stoeckig@mail.uni-mainz.de
yThe cDNA sequence reported in this paper is deposited with Gen-
bank under accession number AJ310148
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00029-9

1732
T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
at 4.63 ppm is characteristic for the b-glucosidic lin-
kages which also identifies the formed arbutin as an
O-b-glucoside.
The simple purification of a crude protein extract based
on one-step chromatography on Ni-nitrilotriacetic acid
(Ni-NTA) delivers AS in very pure form (ꢁ98%), esti-
mated by Coomassie-blue stained SDS-polyacrylamide
gel electrophoresis (SDS–PAGE), which was directly
used for determination of the enzyme properties. The
thermal stability of AS was surprisingly high, as
demonstrated by a temperature optimum of 50 ꢀC. It is
possible to perform assays for 1 h at this temperature
without significant loss of enzyme activity. At 60 ꢀC, the
enzyme shows still 20–50% of its activity depending on
the length of pre-incubation.
Figure 1. Enzymatic formation of arbutin catalyzed by the novel
enzyme arbutin synthase.
In the present paper, we report on the properties of a
recently detected glucosyltransferase from plant cell
suspension cultures of the Indian medicinal plant Rau-
volfia serpentina, catalyzing the biosynthesis of arbutin
(Fig. 1).6 The enzyme is named arbutin synthase (AS)
(EC 2.4.1...) and has been actively expressed in Escher-
ichia coli.7 It is a novel member of the Class IV GTs
belonging to the Nucleotide Recognition Domain type
1b (NRD1b) family of glycosyltransferases.8 The trans-
ferase exhibits the broadest acceptor substrate specifi-
city compared to any of the glucosyl- or
glycosyltransferring enzymes previously described. The
multifunctional nature of AS sets this enzyme apart
from other enzymes of plant secondary metabolism. In
addition based on sequence alignment studies AS occu-
pies a special position within the GT enzyme super
family.
When the pH-dependence of AS was investigated two
optima were measured with nearly the same enzyme
activity, one optimum at pH 4.5, the other at 6.8 in
phosphate buffer (KPi) or 7.5 in citric acid/phosphate
buffer, respectively. To exclude temperature effects we
verified the result by repeating the experiment at 30 ꢀC
(data not shown), which yielded the same results.
Although this feature has been observed for other
enzymes like an endopeptidase from Bacillus inter-
medius10 or phytase from Aspergillus niger11 there is no
rational explanation for this rather unusual phenom-
enon. One possibility is that the two pH optima would
enable the enzyme to function in intracellular compart-
ments where pH can vary, such as the vacuole.12 Beside
the very broad substrate specificity (see below) this
assumption would corroborate the putative physio-
logical role of the enzyme — the detoxification of
harmful phenolics. Since most of the conjugated phen-
olics are stored in vacuoles, a co-localization of the GT
would assure that the enzyme encounters its substrates
and does not interfere with other primary or secondary
pathways, as hypothesized recently.5 However, with the
data obtained so far the subcellular localization of the
enzyme remains unknown and needs further investiga-
tion. It might be noteworthy that other known UDPG
dependent glucosyltransferases from plants exhibit an
optimum between pH 7.5 and 8.513À15 or around pH
9.0.16 Even the luteolin glucuronosyl transferase from
rye, which was detected in the vacuolar fraction17
exhibited an pH-optimum of 7.0.18
Results and Discussion
Arbutin, the O-b-d-glucoside of hydroquinone, is
widely distributed among plants especially of the famil-
ies Ericaceae, Rosaceae and Saxifragaceae. The gluco-
side has slight antibiotic activity based on the release of
the aglycone hydroquinone during its intestinal meta-
bolism. Arbutin also inhibits the melanin biosynthetic
pathway and is therefore applied as a skin lightener in
specific cosmetics.9 Moreover, the compound is one of
the most structurally simple plant-derived glucoconju-
gates. This glucoside is biosynthesised by an UDP-glu-
cose dependent hydroquinone:O-glucosyltransferase
(named arbutin synthase, AS), recently isolated from
cell suspension cultures of the plant R. serpentina (Fig.
1).6 This enzyme belongs to the large enzyme family of
glycosyltransferases (GTs), presently consisting of more
than 200 members.
As previously described, we subcloned the AS cDNA
from Rauvolfia into the pQE60 expression vector and
transferred it into M15 E. coli cells.7 These cells were
used to produce sufficient enzyme for the extensive
characterization presented here, starting with rigorous
identification of the enzyme product by enzymatic and
spectroscopic analyses. The enzyme product was sub-
jected to enzymatic hydrolysis by a- or b-glucosidase;
the latter split the product, arbutin, quantitatively into
hydroquinone and glucose. There was, however, no
hydrolysis observed in presence of the a-glucosidase,
which clearly demonstrates the formed arbutin was a b-
glucoside. The 1H and 13C NMR spectra of the enzyme-
product coincided in every respect with those of com-
mercially available arbutin. The large coupling constant
(7.6 Hz in the 1H NMR spectrum) of the anomeric H-10
To reveal the function of the AS within Rauvolfia cells a
careful and extensive analysis of the endogenous sub-
strate was performed. The occurrence of endogenous
arbutin, together with the low Km (<1 mM) provides
evidence that hydroquinone (also identified by MS ana-
lysis) indeed is the natural substrate for the AS. In con-
trast, many GTs from various plants like Gardenia
jasminoides and Alfalfa exhibit much higher Km values
for their natural substrate,16,19 but have not been
reported to catalyze the reaction of AS. In the 1960s, it
was shown that Vicia faba seedlings and wheat germ
extracts were capable of synthesizing arbutin when
treated with hydroquinone,20,21 but so far no data have
been published regarding the responsible enzyme or the
presence of endogenous arbutin in those plants.

T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
1733
Table 1. Chemical structures of compounds accepted as a substrate by heterologously expressed AS and comparison of relative enzyme activities
R1
R2
R3
R4
%
1. Hydroquinone
–H
–CH3
–OCH3
–H
–OCH3
–CH2OH
–H
–H
–H
–H
–OCH3
–H
–H
–OH
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–OCH3
–H
–H
–OH
–Cl
–OH
–H
–H
–H
–H
–H
–H
–H
–H
–CH3
–H
–H
–H
–H
–H
–H
–H
–H
100
19.0
16.7
10.9
9.4
9.4
8.7
8.6
7.6
7.5
6.2
5.1
2.8
2.2
1.8
1.4
1.2
1.1
1.0
1.0
0.8
0.8
0.2
0.1
2. 4-Chloro-2- methylphenol
3. 2-Methoxy- hydroquinone
4. 3-Methoxyphenol
5. Vanillina
6. Saligenina
7. Resorcinola
8. 4-Hydroxy- benzaldehyde
9. Thymola
10. 4-Chlorophenol
11. 4-Nitrophenol
12. Phenola
13. 4-Methylphenol
14. Methylvanillatea
15. 4-Hydroxyaceto-phenonea
16. 4-Benzyloxy-phenol
17. 4-Hydroxy- thiophenol
18. Biphenyl-2-ol
19. Vanillic acida
20. Carvacrol
21. 4-Methoxy- phenola
22. 3.4-Dimethoxy- phenol
23. Isovanillic acid
24. 4-Hydroxy- benzoic acid
–H
–CHO
–H
–H
–CHO
–H
–Cl
–H
–CH(CH3)2
–H
–H
–H
–H
–OCH3
–H
–H
–NO2
–H
–CH3
–COOCH3
–COCH3
–O–C6H5
–SH
–H
–COOH
–H
–H
–C6H5
–OCH3
–CH3
–H
–H
–OCH3
–H
–H
–H
– CH(CH3)2
–H
–H
–COOH
–H
–OCH3
–OCH3
–H
–COOH
R1
¼CH2
R1
R2
–H
R2
R3
–OCH3
R3
R4
–OH
R4
%
6.2
%
1. Eugenol
1. Coniferyl alcohola
2. trans-Cinnamyl alcohol
3. o-Coumaric acid*
4. Ferulic acida
–CH2OH
–CH2OH
–COOH
–COOH
–H
–H
–OH
–H
–OCH3
–H
–H
–OH
–H
–H
1.2
0.6
0.4
0.1
–OCH3
–OH
R1
R2
R3
R4
R5
%
1. Apigenin
–H
–H
–H
–OH
–H
–OH
–H
–H
–OH
–OH
–H
–OH
–H
–OH
–OH
–OH
–H
–H
–OH
–OH
–OH
–H
–OH
–OH
–OH
5.8
5.4
3.0
2.6
1.0
2. 3-Hydroxyflavone
3. 7-Hydroxyflavone
4. Quercetin
5. Kaempferol
R1
R2
%
1. Umbelliferone
2. Scopoletin
–OH
–OH
–H
–OCH3
3.6
3.0

1734
T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
Table 1 (continued)
aSubstrates, recently described.7
However, the most important property of this novel
enzyme is its exceptionally low substrate specificity
which could be characterized in a simple way by
HPLC analyses. The applied HPLC method is sui-
table to separate all the formed glucosides from the
corresponding aglycones. The difference in retention
times between all substrates and products was
between 1.5 and 2.5 min depending on the structure of
the tested compounds, with the glucosides being the first
to elute, providing a simple test system for glucosylation.
compounds 5-chloro-8-hydroxyquinoline and 4-chloro-
2-methylphenol are well accepted with 57 and 19%
relative activity followed by the natural occurring phe-
nolics vanillin (Km=440 mM, Fig. 2), saligenin, thymol
(ꢁ8%) or eugenol with 6% rel. act. The flavonoids
such as apigenin, kaempferol, quercetin, 3-hydroxy-
and 7-hydroxyflavone can also serve as an acceptor
substrate of the AS in a relative range of ꢁ1–6%.
Moreover, coumarins are glucosylated at about 3% rel.
act. (Km of umbelliferone=550 mM, Fig. 3). Even the
two isoquinoline alkaloids scoulerine and d,l-iso-
corypalmine are glucosylated, which to our knowledge
is the first example of glucosylation of this class of
alkaloids. Surprisingly cinnamyl alcohol is also con-
verted into its glucoside, because of the allylic OH-
group localized in the side chain. This compound and
isatin-3-oxime are the only exceptions from our finding
that exclusively phenolic structures are accepted by AS.
Based on the latter result, it cannot be excluded at this
stage that the AS, besides the known flavanon-O-glu-
cosyltransferases, could also be responsible in vivo for
the biosynthesis of appropriate glucosides of quite dif-
ferent groups of natural products, as indicated by the
determined Km values; especially because the highly
substituted apigenin, kaempferol and quercetin are
converted into several glucosides. As depicted in Figure
4, up to three different glucosides are formed for
instance from quercetin, indicating that the enzyme
obviously exhibits not only a broad substrate accep-
tance but also a low regioselectivity. By LC–MS mea-
surements it could be proved that exclusively mono-
glucosides are formed, for example, scoulerine is glu-
cosylated at both phenolic OH-residues to the corre-
sponding mono-glucosides. Due to this feature, the AS
differs strongly from the betanidin glucosyltransferase
previously described,22 being much more promiscuous
with respect to the accepted hydroxyl groups of the
substrate.
From 74 tested natural and synthetic phenolics and two
cinnamyl alcohols, 45 were glucosylated in a range from
0.1 to 100% relative activity compared to hydro-
quinone. This broad range of acceptor substrates
consist of 24 simple phenolic structures, four phenyl-
propanoid derivatives, two coumarins, two anthraqui-
nones,
a
naphthol, two hydroxyquinolines, five
flavonoids including two flavones and three flavonols,
and two isoquinoline alkaloids of the protoberberine
type. In addition to the more simple phenolics, other
substrates with substantial structural differences to the
natural substrate hydroquinone are still glucosylated in
varying extents (Table 1). For example the synthetic
Enzymes of secondary metabolism such as the catalysts
involved in alkaloid biosynthesis usually exhibit high
specificity for their substrates. In some cases, only a
single substrate is known to be accepted by the enzyme,
Figure 2. Dependence of arbutin synthase activity on concentration of
the acceptor substrate vanillin (linear fit correlation coefficient
R=0.99).

T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
1735
as has been observed for (S)-N-methylcoclaurine-30-
hydroxylase23 in isoquinoline alkaloid biosynthesis or
for polyneuridine aldehyde esterase24 which is a central
enzyme in the formation of certain monoterpenoid
indole alkaloids. Previous work on methyltransferases,
however, points out that this class of enzymes might
have varying methylating capabilities accepting for
instance hydroxylated cinnamic acids and flavonoid
aglycones. Surprisingly, in
a recent investigation
methyltransferases have been shown to act on more
different natural products like simple catechols, phenyl-
propanoids and structurally complex isoquinoline alka-
loids.23,25 This indicates their multifunctional
properties, which is also true for arbutin synthase. Our
current knowledge on the substrate specificity of arbutin
synthase is summarized in Table 1. Although none of
these substrates reached conversion rates comparable to
hydroquinone, the result indicates that the enzyme in
principal tolerates substrates with very diverse chemical
structures, belonging to many classes of plant natural
products. Beside O-methyltransferases, in fact, arbutin
synthase shows the greatest diversity of functions in
plant secondary metabolism for any enzyme currently
characterized.
Although we have, with these experiments, clearly
demonstrated the broad acceptance of a variety of
compounds, a number of specific structural types could
not be converted (Table 2). For instance, carboxy
groups were not transformed into glucose esters, a reac-
tion which is catalyzed by the recently described
hydroxycinnamate glucosyltransferases.26 Methoxylated
cinnamic and a range of benzoic acids such as trimethoxy
cinnamic acid, benzoic acid, acetylsalicylic acid, gallic,
and salicylic acid were not glucosylated. Non-accep-
tance of nicotinic acid, indole-acetic acid and trypto-
phan supports the above observation. Additional
experiments also showed that compounds such as ani-
line, 4-aminophenol or tryptamine will not act as
acceptor substrates for AS, indicating that glucosylation
of amino groups will not occur and that a range of lig-
nans and anthocyanines is not glucosylated. Most
likely, arbutin synthase must be considered as an
enzyme which still recognises specific structural ele-
ments which, however, cannot yet be defined in detail at
this stage of investigation.
Figure 3. Dependence of arbutin synthase activity on concentration of
the substrate umbelliferone (linear fit correlation coefficient R=0.99).
By testing the glucosylation activity of AS in presence of
substances not recognized as a substrate, only dichloro-
phen was found to significantly inhibit the enzymatic
formation of arbutin by the synthase. Further analysis
of this phenomenon revealed dichlorophen to be a
reversible, non-competitive inhibitor (Fig. 5) exhibiting
a Ki of 90 mM, but several substances with related
structural characteristics did not show inhibition of
Figure 4. Formation of several glucosides from the flavonoid querce-
tin catalyzed by arbutin synthase in presence of UDPG. (A) Substrate
(Quercetin) incubated with arbutin synthase; (B) incubated in presence
of enzyme and UDPG; (C) incubation mixture of (B) re-incubated
with 50 nkat b-glucosidase: (1) Quercetin; (2) Imidazole from enzyme
solution; (3) mixture of enzymatically formed quercetin glucosides; (4)
UDP-glucose.
Table 2. Compounds, not accepted by heterologously expressed AS as a substrate
Thiophenol
Catechol
Benzoic acid
Menthol
Tryptophan
Indole-3-acetic acid
Rhapontin, Rhaponticin
2-Demethoxy-thiocolchicin
5-Hydroxy-flavone
Pelargonidin
Aniline
Gallic acid
Salicylic acid
Nicotinic acid
Tryptamine
Morphine
4-Aminophenol
Benzyl alcohol
Acetylsalicylic acid
Dichlorophen
5-Hydroxytryptamine
Sarpagine
Podophyllotoxin
2.4.5-Trimethoxy-cinnamic acid
Malvidin
Resorufin
p-Coumaric acid
Cyanidin
Arctigenin
Pinoresinol

1736
T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
Figure 5. Determination of the inhibition type of dichlorophen and Ki
by using a Dixon plot.
Figure 6. Dependence of arbutin synthase on the concentration of the
donor-substrate UDPG (linear fit correlation coefficient R=0.94).
arbutin formation. We also tested the influence of metal
ions on the reaction catalyzed by AS. In presence of 10
mM of the appropriate salt, Mn2+, Ca2+, Co2+ and
Zn2+ inhibited the arbutin formation, showing 75.3,
75.6, 45.8 and 12.6% of the original activity. Addition
of 10 mM Mg2+ increased the conversion rate to
168.3%. This result is comparable to other known glu-
cosyltransferases.14,16,27 When checking the effect of
Hg2+ we discovered that the enzymatic reaction was
completely inhibited. This inhibition was reversible by
the addition of 20 mM b-mercaptoethanol (65% activi-
tiy recovered). These results suggest that AS has a SH
group near or at the catalytic center, as recently
assumed for a coumarin glucosylating enzyme.27 By
examining the donor substrate specificity it was found
that only pyrimidine nucleotide activated glucose
(TDPG at 68.8% and CDPG at 2.2% compared to
UDPG) was accepted. The purity of CDPG was ver-
yfied to exclude impurities, such as traces of UDPG.
The Km value of UDPG is 77 mM (Km TDPG=1.1 mM)
demonstrating that UDPG occupies the position of the
Figure 7. Nucleotide sequence and deduced amino acid sequence of
AS from R. serpentina. Start and stop codons are marked by ?. Casein
kinase II phosphorylation sites are underlined, protein kinase C phos-
phorylation sites are shaded, ASN-glycosylation site is written white
on black. The small NRD1b-domain is dot-framed, the large NRD1b-
domain is bold-framed.
most preferred donor substrate (Fig. 6). In contrast,
other activated sugars such as UDP-mannose and
UDP-galactose, or UDP-glucuronic acid did not func-
tion as co-factors. Together with the data for other
plant glucosyltransferases, which have been shown to
exclusively accept UDP-glucose as a co-factor,13,14,27À29
this finding is in sharp contrast to the assumed proper-
ties of bacterial glycosyltransferases involved in second-
ary metabolism.30

T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
1737
Figure 8. Dendrogram, constructed by sequence-alignment with ClustalW at the European Bioinformatics Institute server illustrates the phylo-
genetic position of arbutin synthase to different clusters of glucosyltransferases designed recently.4
From the limited amount of data available on GTs in
general, it is, however, extremely difficult to draw final
conclusions about their substrate specificity. In all
available reports on GTs, only a small number of sub-
strates was tested, usually having very similar structures
and belonging to only one class of natural products.
Moreover, in many cases only enriched enzymes were
assayed without a guarantee that no other GTs were co-
purified. Even for heterologously expressed transferases,
no effort was made to obtain a pure enzyme prepara-
tion, a point recently criticized in the literature.5,14 Also,
for the majority of the described plant-derived trans-
ferases identity was infered only in terms of the enzymes
association with the biosynthetic formation of flavonoid
and anthocyanin glucosides.15,16,31À33
AS from Rauvolfia cell suspension cultures.6 The cata-
lyzed reaction of AS proceeds with inversion of the
configuration at the anomeric center of the glucosyl
donor compared to the formed glucoconjugate. There-
fore AS should belong to the NRD1b enzyme family,
representing the inverting GTs.8 This enzyme family is
termed after the ‘Nucleotide Recognition Domain 1b0
which is suspected to be responsible for the recognition
of the UDP or TDP portion of the sugar donor. Not
only the exclusive acceptance of pyrimidine nucleotide
derived donor substrates (UDPG, TDPG, CDPG) but
also a sequence analysis and alignment of AS supports
clearly this classification. Based on an earlier classifica-
tion of GTs8 sequence analysis of AS showed two family
specific sequences, a small NRD1bS domain beginning
in position 266 and the large NRD1bL domain starting
at position 363, both framed in Fig. 7. The sequence also
denotes the presence of Ser at position 365 followed by a
strictly conserved Glu three amino acids downstream,
both amino acids characteristically located in the middle
of the NRD1bL site. The Ser is suggested to be a
potential phosphorylation site,35 which might have a
regulatory function if it would belong to the catalytic
center of the enzyme. However, the catalytic site of all
these sugar-transferring enzymes is not known. There
are several putative phosphorylation sites, as well as a
putative glycosylation site in the whole sequence of AS
but their functional role is so far unknown. Eight amino
acids upstream of Glu 368 a His residue is found, which
again is characteristic for the NRD1b family, and is
eventually involved in the catalyzed reaction as a proton
donator.8 Accepting the published classification of
GTs,8 AS would be a novel member of Class IV of the
NRD1b enzyme super family.
In marked contrast, the arbutin synthase preparations
employed here were pure and the enzyme was rigorously
characterized in terms of substrate acceptance. Jones
and Vogt suggested a classification of GTs, grouping
the six so far best characterized heterologously expres-
sed plant GTs into classes with broad, intermediate, and
narrow substrate specificity.5 Adopting this ranking
arbutin synthase clearly qualifies for the top rank within
the broad substrate specificity group. All results now
obtained seem to place this enzyme in a rather out-
standing position of GTs, which to date have been
assumed to be highly specific, only accepting structural
very related structures.14,31,33,34
In order to gain more detailed insight into the relation-
ship of AS to other GTs on a molecular basis, we ana-
lyzed the nucleotide and amino acid sequence of the AS.
The full-length AS-cDNA clone expressed in E. coli
exhibits an open reading frame of 1413 base pairs (Fig.
7). The peptide sequence deduced from this frame con-
sists of 470 amino acids and corresponds to a calculated
molecular mass of 51.8 kDa which matches well with
the value of 52.5 kDa determined after purification of
A sequence comparison using FASTA3 at the European
Bioinformatics Institute indicated highest identity
[Smith-Waterman score: 62.9% identity (63.5% ungap-
ped)] with a putative UDPG-dependent transferase

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T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
Figure 9. Alignment of deduced amino acid sequences (single-letter code) of AS with most similar glucosyltransferases from plants. The alignment was
created using the ClustalW program at the server of the European Bioinformatics Institute (EBI). The ‘PSPG box’, representing the proposed binding site
of UDP-glucose is framed. Motif A ++++, motif B xxxx, conserved in 95% of all glucosyltransferases ". AS: Arbutin synthase from R. serpentina, AT:
putative Flavonol-glucosyltransferase from Arabidopsis thaliana (CAB80916), CGT1: UDP-glucosyltransferase from Manihot esculenta (X77459),
CGT4: UTP-glucosyltransferase from Manihot esculenta (X77462), SB: UDP-glucosyltransferase from Solanum berthaultii (AF006081).
(Q9M156) identified in the Arabidopsis genome project.
The function of the latter enzyme needs, however, to be
determined.
nus, is highly characteristic for those glycosyltrans-
ferases responsible for the formation of natural product
glycosides. The two highly conserved motives A and B
found in this box (WAPQV and HCGWNS, respec-
tively) for which WQ and HGS can be identified in
ꢁ95% of all b-GTs4 are again evidence for the correct
classification made here for arbutin synthase.
A multiple alignment using sequences of thirty three
NRD1b GTs places AS between enzyme clusters II and
III of a phylogenetic dendrogram (Fig. 8) created very
recently.4 Based on the presently available data, this
result points to an outstanding position of the AS. Four
sequences of GTs most similar to AS and placed as well
between cluster II and III, were aligned with that of AS.
As illustrated in Figure 9 the framed consensus
sequence named ‘plant secondary product GT’ (PSPG-
box)36 representing the assumed binding site of UDPG
can be realized. This sequence always near the C-termi-
Conclusion and Prospect
The heterologous expression, characterization, and
molecular analysis of the novel arbutin synthase, which
is not only a new member of Class IV of NRD1b gly-
cosyltransferases, but obviously also the most multi-

T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
1739
functional enzyme of plant secondary metabolism
known up to date, provides the major prerequisite to
understand in greater detail the still unknown catalytic
mechanism of this enzyme class. Site-directed mutagen-
esis will now permit identification of the proposed
binding sites, the catalytic domains and the single amino
acid residues which have been suggested as candidates
of the catalytic center of GTs. Most probably mutant
enzymes might be generated with significantly extended
substrate acceptance, which could be used for efficient
— also technical — production of structurally diverse
and novel glucosides. Because AS is also easily available
in a higher mg-scale X-ray structure analysis and/or
high field NMR structure determination might become
possible in the near future and would be the first exam-
ple to clarify the three-dimensional structure of an
eucaryotic enzyme of the NRD family. Appropriate
experiments are now in progress.
trile and 98% water, pH 2.3 (H3PO4); UDPG 1.2 min,
arbutin 3.2 min, and hydroquinone 4.2 min.
Properties of the arbutin synthase
To determine the temperature optimum of AS, the
standard activity assay was used (20 min incubation
time) but incubation temperature varied from 5 to
90 ꢀC. Before starting the reaction, the enzyme solution
and the other components were pre-incubated at the
desired temperature for 5, 10 and 15 min. The reaction
was started by mixing all components. The pH-depen-
dence of the transferase was measured at 30 and 50 ꢀC
using buffer in a range from pH 2–9: citrate/NaOH
buffer pH 2–6, phosphate buffer pH 5–9. In addition,
citric acid/phosphate buffer was tested in a pH range
from 2.2 to 8. Incubations were performed for 20 min.
For the measurements of the acceptor substrate specifi-
city the standard assay was employed replacing hydro-
quinone with the putative substrates. After addition of
25 pkat AS the mixture was incubated at 50 ꢀC for 40
min. The enzyme was precipitated with 300 mL MeOH,
centrifuged at 18,000g for 5 min, and the supernatant
analyzed by HPLC to assess conversion rates. A linear
gradient program starting with 2% acetonitrile and
98% water, pH 2.3 (H3PO4), and ending after 8 min
with 80% acetonitrile and 20% water was employed.
Determination of enzyme specificities regarding the
donor substrate was performed by incubating the sub-
stances (UDPG, TDPG, CDPG, GDPG and UDP-
mannose, UDP-galactose, UDP-glucuronic acid) in the
standard assay described above. After addition of 300
mL MeOH the supernatant was analyzed by HPLC.
Purity of CDPG was checked by TLC using an EtOH/
H2O (85/15+0.1H3PO4) solvent system, and thymol
reagent (0.5 g thymol, 5 mL 96% H2SO4, 95 mL 96%
EtOH); Rf of CDPG=0.44, UDPG=0.5) with UV (254
nm) detection. For checking the influence of metal ions
on the activity of AS, standard assays were used with
addition of 10 mM (final concentration) of the desired
ions. Salts used were MnSO4, CaCl2, CoCl2, MgCl2,
ZnSO4 and HgCl2. Examination of the SH dependence
of arbutin synthase was performed by adding 10 mM
HgCl2 to a solution containing 65 pkat enzyme. After
testing the activity (as described above) BME to a final
concentration of 20 mM was added and arbutin forma-
tion was checked by HPLC. For determination of the
inhibition type of dichlorophen and of the inhibition
constant, variable concentrations of dichlorophen
(0.001–0.1 mM) were incubated with 180 pkat AS in the
presence of 1 mM HQ, 2 mM UDPG and 100 mM Tris–
HCl, pH 7.5 in a total volume of 127.6 mL for 10 min at
50 ꢀC. After precipitating the enzyme with 300 mL
MeOH, the supernatant was analyzed as described above.
Experimental
Purification of His-tagged AS
Recombinant E. coli were grown for 24 h at 25 ꢀC and
100 rpm in 24 1-L Erlenmeyer flasks containing 400 mL
Luria-Bertani (LB) nutrition medium each [100 mg/L
ampicillin, 25 mg/L kanamycin, isopropyl-b-d-thio-
galactopyranoside (IPTG) 0.3 mM]. At an OD600 of 0.9,
the bacteria were harvested by centrifugation at
11,000g. A pellet of 15 g bacteria was resuspended in
250 mL buffer [KPi 50 mM, pH 8.0, 300 mM NaCl, 10
mM imidazole and 20 mM b-mercaptoethanol (BME)]
and 1 mg/mL lysozyme was added. After 30 min incu-
bation on ice, the cells were lyzed by sonication (75 W, 6
ꢂ 10 s). The lysate was centrifuged at 10,000g for 30
min and the enzyme-containing supernatant was
pumped through a Ni-nitrilotriacetic acid (Ni-NTA)
column (volume 2 mL) at a flow rate of 0.5 mL/min.
The column was washed with 20 column volumes of
buffer with 20 mM imidazole. Elution was achieved
with an imidazole gradient (20–250 mM). The purity of
AS was demonstrated by Coomassie-blue stained SDS-
PAGE.
Protein determination
Enzyme concentrations were measured as described by
Bradford.37 The procedure was the same as recently
published.6
Activity assay for AS
For monitoring the transferase activity variable enzyme
amounts were added to an aqueous solution containing
1 mM hydroquinone, 2 mM UDPG, 100 mM Tris–HCl,
pH 7.5 in a total assay volume of 63.8 mL (standard
assay). After incubating at 50 ꢀC, for different times, 300
mL MeOH was added and the mixture centrifuged at
18,000g for 5 min. The supernatant was subjected to
HPLC analysis, using a 250 ꢂ 4 mm LiChrospher 60
RP-select B column (5 mm) (Merck, Darmstadt, Ger-
many) and a solvent system consisting of 2% acetoni-
Identification of products from the AS-reaction
To verify the formation of b-glucosides, the MeOH
added to the incubation mixture was removed by eva-
poration and 100 mL of a solution, containing 50 nkat
almond-derived b-glucosidase (Sigma, Deisenhofen,
Germany) in citrate buffer (100 mM, pH 5.0) was
added. This mixture was incubated at 37 ꢀC for 3 h. The

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T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
1
reaction was terminated with 300 mL MeOH followed
by centrifugation (5 min, 18,000g). The supernatant was
analyzed by the above described HPLC method. Enzy-
matically formed arbutin (0.125 mmol) was incubated
for 5 h at 37 ꢀC in presence of b-glucosidase (20 nkat)
and a-glucosidase (20 nkat) from brewers yeast (Sigma,
Deisenhofen, Germany) in 100 mM citrate buffer (pH
5.0) and 100 mM KPi (pH 6.0), respectively. The reac-
tion was stopped by addition of 300 mL MeOH and
centrifuged at 18,000g (5 min). The supernatant was
analyzed by HPLC. Multiple b-glucoside formation: In
a total volume of 128 mL Tris–HCl (100 mM, pH 7.5)
quercetin and scoulerine, each (0.125 mmol), UDPG
(0.25 mmol) and 200 pkat arbutin synthase reacted for 5
h at 37 ꢀC. After addition of MeOH (300 mL) and cen-
trifugation (18,000g), 50 mL of the supernatant was
subjected to HPLC analysis. MeOH was evaporated
and to the remaining incubation mixture b-glucosidase
(50 nkat in 150 mL citrate buffer 100 mM, pH 5.0) was
added and incubated at 37 ꢀC for 5 h. After termination
of the enzymatic reaction with 300 mL MeOH and cen-
trifugation, HPLC analysis was performed. For deter-
mination whether different mono-glucosides or multiple
glucosylated products are formed by AS the same assay
was used. After terminating the reaction as described
above, the complete mixture was freeze-dried. The resi-
due was dissolved in 500 mL MeOH. This solution was
subjected to LC–MS measurements on a PE SCIEX
API 165 instrument (Perkin–Elmer, Langen, Germany).
Column used was a 250 ꢂ 4 Select B (Merck, Darm-
stadt, Germany) and a linear gradient program, starting
with 15% of MeOH/acetonitrile with 5 mM ammonium
formate (1:1) (system A) and 85% 5 mM ammonium
formate buffer, pH 4.0 (system B) and ending after 30
min with 100% of system A at a flow of 0.9 mL/min.
MS conditions: electrospray ionization (ESI) positive
and negative. Quercetin mono-glucoside (1) 11.7 min
[m/z (À)-ESI 463; quercetin minus glucose (m/z (À)-ESI
301)], quercetin mono-glucoside (2) 12.6 min [m/z (À)-
ESI 463; quercetin minus glucose (m/z (À)-ESI 301)];
scoulerine mono-glucoside (1) 6.9 min [m/z (+)-ESI
490, (À)-ESI 488; scoulerine minus glucose (m/z (+)-
ESI 328, (À)-ESI 326)], scoulerine mono-glucoside (2)
8.4 min [m/z (+)-ESI 490, (À)-ESI 488; scoulerine
minus glucose (m/z (+)-ESI 328, (À)-ESI 326)]. Further
identifications were done by NMR or EI-mass spectro-
Vanillinglucoside. H NMR (D2O, 300 MHz): d (ppm)
9.86 (1H, s, H-7); 7.69 (1H, dd, J=1.8, 8.3 Hz, H-6), 7.6
(1H, s, H-2), 7.40 (1H, d, J=8.3 Hz, H-5), 5.36 (1H, d,
J=7.3 Hz, H-1), 4.01 (3H, s, H-8), 4.00 (1H, dd, partly
overlapped by H-8, J=1.8, 12.2 Hz, H-6A0), 3.86 (1H,
dd, J=5.5, 12.2 Hz, H-6B0), 3,77–3.56 (4H, m, H-20-H-
50). 13C NMR (75 MHz, D2O): d [ppm] 195.7 (C-7),
152.1 (C-4), 149.9 (C-3), 132.0 (C-1), 127.6 (C-6), 115.8
(C-5), 112.4 (C-2), 100.5 (C-10), 77.2 (C-30), 76.3 (C-50),
73.5 (C-20), 70.1 (C-40), 61.3 (C-60), 56.8 (C-8). Tetra-
acetyl-vanillinglucoside; EI–MS, m/z (rel.int.%): 331
(7), 271 (4), 229 (2), 211 (3), 169 (93), 152 (15), 137 (2),
127 (30), 109 (100 ), 97 (14), 81 (12).
Endogenous substrate of AS
To trace indirectly the endogenous substrate of arbutin
synthase, hydroquinone, cell cultures from R. serpentina
were analyzed for the occurrence of arbutin. Fresh cells
of 230 g wet weight were lyophylized resulting in 9 g dry
material. The obtained powder was extracted over night
at 100 rpm with different solvents [petrolether, 400 mL;
ether/MeOH (1:1), 400 mL; MeOH, 400 mL]. The pet-
rolether extract was rejected. The ether/MeOH and
MeOH extracts were combined and freeze-dried after
solvent evaporation. The residue (1 g) was dissolved in
100 mL water and purified over a XAD-column (80 mL
volume). After elution with MeOH (300 mL) and con-
centration under vacuum, the solution was fractionated
(12 mL per fraction) using a 250 ꢂ 10 mm LiChrospher
100 RP-18 column (Merck, Darmstadt, Germany) [lin-
ear gradient program starting with 2% MeOH/H2O
(8:2) and 98% H2O (0.05 v/v trifluoroacetic acid) and
ending after 30 min with MeOH/H2O (8:2)]. To 250 mL
of selected fractions 250 nkat almond-derived b-gluco-
sidase (Sigma, Deisenhofen, Germany) in 250 mL citrate
buffer (100 mM, pH 5.0) was added. After 4 h at 37 ꢀC
the reaction was stopped with 500 mL MeOH and cen-
trifuged at 18,000g for 5 min. HPLC analysis of this
solution did not show the presence of arbutin. The
supernatant was freeze-dried and after dissolving of the
residue in 200 mL water 200 pkat of arbutin synthase
and 10 mL 25 mM UDPG were added and incubated for
4 h at 50 ꢀC. After terminating the reaction with 500 mL
MeOH and subsequent centrifugation the supernatant
was subjected to HPLC analysis. A peak at 3.1 min
indicated the presence of arbutin. For further identifi-
cation chromatography on two TLC plates (Silica gel 60
F254, Merck, Darmstadt, Germany) was performed;
solvent system: EtOAc/CHCl3/HCOOH/H2O — 60:19:
12:9. The resulting spot (Rf=0.44) on the plate localized
by UV (254 nm) had the same Rf value as arbutin. The
spot was eluted with 20 mL CH2Cl2/MeOH (1:1) and the
solvent evaporated. The residue was dissolved in 50 mL
H2O, one fraction was incubated with 83 pkat b-gluco-
sidase in 150 mL of the above citrate buffer for 1 h and
analyzed by the above described TLC system, Rf=0.88
(identical to hydroquinone reference). Another part was
acetylated with pyridine/acetic anhydride (1:1) after
treatment with b-glucosidase, dried and subjected to
MS-analysis: Diacetyl-hydroquinone; EI–MS, m/z
(rel.int.%): 194 (4), 152 (19), 110 (100), 81 (10). A sam-
ple of authentic arbutin and the eluate of the spot
1
metry. H and 13C NMR spectra were obtained using
either a Bruker (Karlsruhe, Germany) ARX 400 or AC
300 instrument. Solvents used were D2O or DMSO-d6.
MS spectra were measured on a Finnigan MAT (Bre-
men, Germany) type MAT 44 S instrument at 70 eV.
1
Arbutin. H NMR (DMSO-d6/D2O, 400 MHz): d (ppm)
6.86 (2H, d, J=9.2 Hz, H-2, H-6), 6.65 (2H, d, J=9.2
Hz, H-3, H-5), 4.63 (1H, d, J=7.6 Hz, H-10), 3,67 (1H,
dd, J=1.8, 12.0 Hz, H-6A), 3.44 (1H, dd, J=5.9, 12.0,
H-6B), 3.23–3.08 (4H, m, H-20-H-50). 13C N MR
(100 MHz, DMSO-d6): d (ppm) 152.2 (C-1), 150.3 (C-4),
117.7 (C-2, C-6), 115.6 (C-3, C-5), 101.8 (C-10). 76.9 (C-
30), 76.6 (C-50), 73.3 (C-20), 69.8 (C-40), 60.8 (C-60).
Pentaacetyl-arbutin; EI–MS, m/z (rel. int.%): 331 (6),
271 (3), 229 (5), 169 (82), 139 (10), 127 (32), 109 (100),
97 (19), 81 (19).

T. Hefner et al. / Bioorg. Med. Chem. 10 (2002) 1731–1741
1741
(Rf=0.44, described above) were also acetylated and
measured by MS. The obtained spectra were identical;
EI–MS, m/z (rel. int.%): 331 (6), 271 (3), 229 (5), 169
(82), 139 (10), 127 (32), 109 (100), 97 (19), 81 (19).
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Acknowledgements
Financial support provided by the Deutsche For-
schungsgemeinschaft (Bonn-Bad Godesberg) and by the
Fonds der Chemischen Industrie (Frankfurt/Main,
Germany) is very much appreciated. We also thank
Mark Smith (Dept. of Chemical Engineering, Cornell
University) for verifying the English version of the
manuscript and Christiane Hinse, Irina Gerasimenko
(Mainz, Germany) for spectroscopic analyses.
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