Probingsuggestedcatalyticdomainsof glycosyltransferases
by site-directed mutagenesis
Tobias HefnerandJoachim Sto¨ ckigt
Lehrstuhl fu
¨
r Pharmazeutische Biologie, Johannes Gutenberg-Universita
¨
t Mainz, Germany
The plant enzyme arbutin synthase isolated from cell sus-
pension cultures of Rauvolfia serpentina and heterologously
expressed in Escherichia coli is a member of the NRD1b
family of glycosyltransferases. This enzyme was used to
prove, bysite-directed mutagenesis, suggested catalytic
domains and reaction mechanisms proposed for enzyme-
catalyzed glycosylation. Replacement of amino acids far
from the NRD domain do not significantly affect arbutin
synthase activity. Exchange of amino acids at the NRD site
leads to a decrease of enzymatic activity, e.g. substitution of
Glu368 by Asp. Glu368, which is a conserved amino acid in
glycosyltransferases located at position 2 and is important
for enzyme activity, does not serve as the nucleophile in the
catalytic centre as proposed. When it is replaced by Ala, the
resulting mutant enzyme E368A exhibits comparable acti-
vity as found for E368D in respect to vanillin. Enzyme
activities of wild-type and E368A towards several substrates
were not affected at the same level. His360 at position 1 of
NRD1b glycosyltransferases occupies a more crucial role as
expected. When it is exchanged against other basic amino
acids such as Lys or Arg the enzyme activity decreases
1000-fold. Replacement of His360 by Glu leads to a
mutant enzyme (H360E) with an 4000-fold lower activity
compared with the wild-type. This mutein still produces a
b-glucoside, not an a-glucoside and therefore indicates that
generation of the typical E–E motif of NRD1a glycosyl-
transferases does not convert a NRD1b enzyme into a
NRD1a enzyme. The presented data do not support several
suggestions made in the literature about catalytic amino
acids involved in the glycosyltransfer reaction.
Keywords: arbutin synthase; catalytic domains; NRD
glycosyltransferases; reaction mechanism; site-directed
mutagenesis.
The transfer of a monosaccharide moiety from an activated
sugar donor to monomeric and polymeric acceptor mole-
cules is a common reaction in nature. The glycosylation of
an enormous variety of natural compounds and also of a
broad range of xenobiotics is catalyzed by more than 300
known glycosyltransferases identified from human, animal,
microbial and plant sources [1,2].
Although some of these transferases have already been
exhaustively investigated for many decades the molecular
mechanism of their action including details of their catalytic
domains remains mostly unexplored. A rapidly growing
amount of sequence data of these enzymes and numerous
sequence alignment studies provide first insights into the
process of glycosylation. They also deliver working hypo-
theses, which might help to establish a better understanding
of these processes although the end conclusions based on
sequence alignments are still highly speculative.
Application of additional approaches such as site-direc-
ted mutagenesisand X-ray analyzes must have priority in
order to solve the catalytic mechanism of glucosyl transfer
in the near future. Heterologous expression of glucosyl-
transferases will be, however, a prerequisite to succeed in
this research.
We have recently isolated a novel glucosyltransferase
catalyzing the glucosylation of hydroquinone from cell
suspension cultures of the Indian medicinal plant Rauvolfia
serpentina Benth. ex Kurz [3]. We named this enzyme
arbutin synthase. Functional heterologous expression of
this synthase in Escherichia coli by the approach of Ôreverse
geneticsÕ allowedustousetheenzymeasoneofthemost
promising candidates to prove general suggestions made
recently on the reaction mechanism of glycosyltransferases.
In this paper we report on appropriate site-directed
mutagenesis experiments performed on arbutin synthase,
which are applied to evaluate the validity of general
mechanistic models of glucose transfer.
Materials and methods
Site directed mutagenesis
Mutagenesis of arbutin synthase (AS) was achieved using
the QuickChange
TM
Site-Directed Mutagenesis Kit (Strat-
agene, La Jolla, USA). As template for PCR AS-pQE60
[¼ AS(His)
6
] construct and the following primer pairs were
used (substituted amino acids are underlined): L(86)Ifor:
Correspondence to J. Sto
¨
ckigt, Department of Pharmaceutical Biology,
Institute of Pharmacy, Johannes Gutenberg-University Mainz,
Staudinger Weg 5, 55099 Mainz, Germany.
Fax: + 49 6131 3923752, Tel.: + 49 6131 3925751,
E-mail: stoeckig@mail.uni-mainz.de
Abbreviations: AS, arbutin synthase; NRD, nucleotide recognition
domain.
Enzymes: arbutin synthase (EC 2.4.1.218).
Note: The cDNA sequence of arbutin synthase from Rauvolfia
serpentina wassubmittedtoGenBankwithaccessionnumber
AJ310148.
Note: Dedicated to Professor Zenk on his seventieth birthday.
(Received 15 April 2002, revised 27 September 2002,
accepted 2 December 2002)
Eur. J. Biochem. 270, 533–538 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03409.x
5¢-GACCCGTATTTGTATCACCATCACTCGCTCTCT
CCCG-3¢, L(86)Irev: 5¢-CGGGAGAGAGCGAGTGAT
GGT
GATACAAATACGGGTC-3¢; A(204)Vfor: 5¢-GGC
CAAGAGATACCGGTTA
GTTGAGGGTATCATGG-
3¢, A(204)Vrev: 5¢-CCATGATACCCTC
AACTAACCGG
TATCTCTTGGCC-3¢; E(368)Dfor: 5¢-GGAACTCT
ATTCTT
GACAGTGTAGTTAATGGGGTGCCG-3¢;
E(368)Drev: 5¢-CGGCACCCCATTAACTACACT
GTC
AAGAATAGAGTTCC-3¢; E(368)A: 5¢-GGAACTCT
ATTCTT
GCGAGTGTAGTTAATGGGGTGCCG-3¢;
E(368)Arev: 5¢-CGGCACCCCATTAACTACACT
CGCA
AGAATAGAGTTCC-3¢; H(360)Rfor: 5¢-GGGTGGATT
TCTAACC
CGATGCGGGTGGAAC-3¢; H(360)Rrev: 5¢-
GTTCCACCCGCA
TCGGGTTAGAAATCCACCC-3¢;
H(360)Kfor: 5¢-CGGGTGGATTTCTAACC
AAGTGCG
GGTGGAAC-3¢; H(360)Krev: 5¢-GTTCCACCCGCA
CTTGGTTAGAAATCCACCC-3¢; H(360)Efor: 5¢-CG
GGTGGATTTCTAACC
GAGTGCGGGTGGAAC-3¢;
H(360)Erev: 5¢-GTTCCACCCGCA
CTCGGTTAGAAA
TCCACCC-3¢. The resulting plasmids were transformed
into E. coli TOP10 and sequenced after purification using
the Nucleo Spin Plasmid Kit (Macherey-Nagel, Du
¨
ren,
Germany).
Protein expression
For expression of the mutant enzymes the plasmids were
transformed into E. coli M15 cells. Cultures in 100 mL LB
(Luria–Bertani) medium, containing 100 mgÆL
)1
ampicillin
and 25 mgÆL
)1
kanamycin, were grown overnight at
200 r.p.m and 37 °C. These cultures which were used to
inoculate 2 L LB medium (antibiotics as before plus 0.3 m
M
IPTG), were cultivated at 100 r.p.m and 25 °C. After 24 h
the cells were harvested by centrifugation at 4000 g for
10 min. The resulting pellets were resuspended in 50 mL
buffer (K
2
HPO
4
50 m
M
,pH8.0,300m
M
NaCl, 10 m
M
imidazole and 20 m
M
b-mercaptoethanol) and 1 mgÆmL
)1
lysozyme was added. After incubation on ice for 30 min the
cells were lyzed by sonification (70 W, 6 · 10 s) and
centrifuged at 12 000 g for 30 min. The resulting superna-
tants were pumped through Ni-nitrilotriacetic acid (Qiagen,
Hilden, Germany) columns (each 1 mL volume) at a flow
rate of 0.5 mLÆmin
)1
. After washing the column with buffer
containing 20 m
M
imidazole the enzyme was eluted by a
linear gradient (20–250 m
M
imidazole) over 20 column
volumes. The purity of the eluted enzymes was checked by
Coomassie-blue stained SDS/PAGE [4].
Protein concentration and activity
Protein concentrations were measured using the method of
Bradford [5] and a standard curve derived from bovine
serum albumin. For testing the activity and determining the
kinetic parameters of arbutin synthase wild-type and
mutant enzymes the following assay was used. A solution
of 1 m
M
hydroquinone or the substrates tested (Fig. 5),
2m
M
UDP-Glu, 100 m
M
Tris/HCl, pH 7.5 in a total
volume of 127.6 lL was prepared. Enzyme in different
amounts was added to this solution and the mixture was
incubated at 50 °C for various times. After terminating the
enzymatic reaction with 300 lL MeOH and centrifugation
at 18 000 g for 5 min the supernatant was analyzed by
HPLC. A 250 · 4 mm LiChrospher 60 RP-select B column
(5 lm) (Merck, Darmstadt, Germany) was used and a
solvent system consisting of 2% acetonitrile and 98% water,
pH 2.3 (H
3
PO
4
). For verifying whether a-glucosides or
b-glucosides were formed with the mutein H360E, an assay
with the following conditions was applied: 1 mg hydroqui-
none, 10 mg UDPG, 50 lg enzyme in 550 lLwater,
containing 100 m
M
Tris, pH 7.5 and 20 m
M
mercaptoeth-
anol was prepared. After incubation at 37 °Cfor17h,the
reaction was terminated with 300 lL MeOH, centrifuged
and freeze-dried. The residue was dissolved in 500 lL
MeOH/H
2
O (7 : 3) and applied to a TLC plate (Silica gel 60
F
254
, solvent system EtOAc/MeOH/H
2
O (7:2:1)). The
bands identified as the glucoconjugates were scratched out
and eluted with 1.5 mL CH
2
Cl
2
/MeOH (7 : 3). Ten micro-
litres of this fraction were mixed with 190 lLMeOHand
analyzed by the above described HPLC method. A peak at
3.6 min clearly showed glucosylated hydroquinone. The
samples were freeze-dried and dissolved in 100 lLH
2
O. To
25 lL of this solution 175 lL citrate buffer (100 m
M
,
pH 5.0) containing 20 nkat almond-derived b-glucosidase
(Sigma, Deisenhofen, Germany) or a-glucosidase (20 nkat)
from brewers yeast (Sigma, Deisenhofen, Germany) in
100 m
M
K
2
HPO
4
(pH 6.0) were added. After incubation at
37 °C for 1 h the reaction was terminated with 300 lL
MeOH followed by centrifugation (5 min, 18 000 g). The
supernatant was analyzed by the HPLC and TLC methods
described above.
Results and discussion
In our previous studies we have described the isolation from
plant cell suspension cultures of R. serpentina a UDP-
glucose dependent enzyme which glucosylates hydroqui-
none with formation of the O-b-
D
-glucoside arbutin
(Fig. 1). Arbutin synthase has also been heterologously
expressedinanactiveforminE. coli [6] followed by a
detailed sequence analysis and investigation of the enzyme
properties, especially of its substrate specificity [7]. Based on
these substrate studies, arbutin synthase is not only a
glycosyltransferase with an exceptionally broad substrate
acceptance but also in this respect exceeds all the so far
known proteins of this particular enzyme family. Indeed it is
an unique enzyme which at the present time exhibits the
most multifunctional character in the metabolism of natural
compounds by converting members of many different
groups of natural products, e.g. phenyl-propanoids, cou-
marins, anthraquinones, flavonoids and protoberberines. In
addition this glucosyltransferase also glucosylates a large
number of phenolic xenobiotics.
In general, all the glycosyltransferases belong to only two
types of enzymes, transferases retaining the stereochemistry
at the anomeric carbon or those inverting the configuration
Fig. 1. Catalyzed reaction of arbutin synthase (AS) isolated from cell
suspension cultures of Rauvolfia s erpentina or heterologously expressed
in E. coli.
534 T. Hefnerand J. Sto
¨
ckigt (Eur. J. Biochem. 270) Ó FEBS 2003
at that centre during sugar transfer [8–10]. Extensive
computer alignments of the amino acid sequences of these
enzymes were used in the past not only for further
classification but also to propose catalyticdomainsof these
proteins and the nature of involved reaction mechanisms.
When applying the approach of Campbell et al.[1,2],who
divided the glycosyltransferases into 26 families, we could
place arbutin synthase clearly in family 1, which consists of
transferases from viruses, bacteria, fungi, higher plants and
animals. The same result was obtained when we followed
the classification of the Cazy-Server (http://afmb.cnrs-mrs.
fr/cazy/CAZY/index.html), subdividing the glycosyltrans-
ferases into 56 different families based on the system of
Campbell et al. [1,2]. Other authors classify glycosyltrans-
ferases after the appearance of the so-called DxD-motif
[11–15], which is believed to be involved in binding the UDP
moiety. In the sequence of arbutin synthase this motif could
not be unambiguously identified. Although there are several
sequences, which could be potential candidates (Fig. 2), the
surrounding amino acids at these sites do not fit an extended
DxD-motif taking into account the properties of neigh-
bouring amino acids. Using hydrophobic cluster analysis
(http://smi.snv.jussieu.fr/hca/hca-form.html) appropriate
clusters, which seem to be important for a typical DxD-
motif [11,15], could not be detected in arbutin synthase
(data not shown). Based on an overwhelming amount of
data of their primary structures, which derive from cloning
of the appropriate cDNAs, the glycosyltransferases are
grouped into NRD1 and NRD2 proteins because of their
nucleotide-recognition-domain. The NRD1 family is fur-
ther classified into the NRD1a and NRD1b subgroups,
depending on the stereochemical course of glycosylation,
which can proceed with retention or inversion of the
stereochemistry at the anomeric centre of the glucose
moiety. The inverting enzymes (NRD1b and NRD2) were
further subdivided into a class showing the motif of a His
(or Arg/Asp) representing position 1 which in general is
located eight amino acids upstream from a Glu residue
(position 2) [8].
As previously classified, most of the NRD1b H(R/K)-E
domain-containing transferases form one specific family.
This family is well separated from another, exhibiting
instead the E-E motif [1,2].
The resulting O-b)glucosides of arbutin synthase but also
conserved sequences such as the small NRD1bSandlarge
NRD1bL domains (Fig. 2) clearly place the enzyme into the
NRD1b family. In agreement with this classification,
arbutin synthase exhibits the His-Glu site (Fig. 2), proposed
as a catalytic domain of general importance for the
mechanism of sugar transfer. The significance of this His-
Glu site we also explored by an alignment based on amino
acid sequences of 24 plant-derived glucosyltransferases
including arbutin synthase. As illustrated in Fig. 3 this
motif is, in fact, completely conserved within the NRD
domain which is a part of the so called plant secondary
product glucosyltransferase (PSPG)-box [9,10]. Therefore
arbutin synthase became an interesting example for the
evaluation of earlier suggestions concerning catalytic amino
acids and proposed reaction mechanisms of glycosyltrans-
ferases [8]. Because site-directedmutagenesis studies have
not so far been performed for eucaryotic glycosyltrans-
ferases in order to prove their catalytic amino acids, we have
generated for this study wild-type arbutin synthase-(His)
6
and seven mutant enzymes of it. These enzymes were
expressed in E. coli M15 strain using the expression vector
pQE-60. Purification of the muteins was facilitated by
introducing a His-tag onto the C-terminus and linear-
gradient elution with imidazole from Ni
2+
-nitrilotriacetic
acid columns. Based on Coomassie-blue staining all these
mutant enzymes showed high purity (Fig. 4). Because the
K
m
value of the natural substrate of the synthase, hydro-
quinone, is extremely small (< 1 l
M
) and very difficult to
measure, we used the substrate vanillin for the determin-
ation of kinetic parameters and enzyme activity (K
m
, V
max
,
k
cat
, k
cat
/K
m
) of the muteins. But the specific enzyme activity
could still be determined with hydroquinone as substrate.
As control mutations we replaced some amino acids which
were not discussed in the literature as important for enzyme
activity and were also far from the NRDs and the His-Glu
domains; Lys86 was changed to Ile and Ala204 to Val
(Fig. 2, Table 1).
The results indicated that enzyme activity was not
drastically influenced by these replacements, probably
because the mutations are far from the nucleotide recogni-
tion sites. Exchange of Lys86 against the neutral Ile resulted
in only a slight increase of the K
m
-value and approximately
18% decrease of V
max
. Replacement of Ala204 to Val
caused a greater decrease of the catalytic efficiency (k
cat
/K
m
),
i.e. approximately threefold. The specific activity of this
Fig. 2. Arbutin synthase amino acid sequence showing the NRD1bS
(boxed) and NRD1bL (dot boxed) domains, putative DxD motifs
(written white on black), and the H–E site in position 1 and position 2
(marked with arrows).
Ó FEBS 2003 Probingcatalyticdomainsof NRD glycosyltransferases (Eur. J. Biochem. 270) 535
mutant enzyme compared with the wild-type was still
between 55 and 60% for the substrates, hydroquinone and
vanillin.
The following mutations did, however, give more intrigu-
ing results. As recently suggested, conserved glutamic acids
of the NRD region located at position 1 and 2 may occupy
the role ofcatalytic residues for those glycosyltransferase
reactions which proceed with retention of the sugar donor
configuration (NRD1a family). Transferases catalyzing the
inverting reaction (NRD1b family) are, however, characte-
rized by His instead of Glu in position 1. In arbutin synthase
this histidine is identified as His360 and the glutamic acid at
position 2 as Glu368.
Provided that the model of transferase mechanisms
proposed in the literature is correct, substitution of Glu in
position 2 for Asp in any member of the NRD1a or
NRD1b family should result in a dramatic reduction in the
reaction rate of such a mutein [8]. This suggestion is due to
the assumption that in both reactions the Glu residue acts as
Fig. 3. Sequence alignment, showing the PSPG-box [9,10], prepared
with 24 plant-derived glucosyltransferase sequences. The alignment was
created using the
CLUSTALW
program at the server of the EBI. *, homo-
logous amino acids; Ô:Õ, conserved substitutions have been observed.
RS: arbutin synthase (R. serpentina, AJ310148), AT I: putative gluco-
syltransferase (GENE:AT2G23260) (A. thaliana, O22182), AT II:
putative glucosyltransferase (GENE:AT2G23250) (A. thaliana,
O22183), AT III: putative glucosyltransferase (GENE:AT2G23210)
(A. thaliana, O22186), DB: betanidin 6-O-glucosyltransferase (D. bel-
lidiformis, Q8W237), FI: flavonoid 3-O-glucosyltransferase (GENE:
UFGT) (F. intermedia, Q9XF16), GM: putative glucosyltransferase
(G. max, Q8S3B7), GT: flavonol 3-O-glucosyltransferase (G. triflora,
Q96493), HV: flavonol 3-O-glucosyltransferase (H. vulgare), LE:
putative glucosyltransferase (L. esculentum, Q8RXA4), ME: flavonol
3-O-glucosyltransferase 1 (M. esculenta, Q40284), NT I: glucosyl-
transferase NTGT2 (N. tabacum, Q8RU71), NT II: UDP-glucose:
salicylic acid glucosyltransferase (N. tabacum, Q9M6E7), PA: Gluco-
syltransferase-14 (GENE:ADGT-14) (P. angularis, Q8S995), PF I:
flavonoid 3-O-glucosyltransferase (P. frutescens, O04114), PF II:
UDP-glucose:anthocyanin 5-O-glucosyltransferase (P. frutescens,
Q9ZR27), PH: anthocyanin 5-O-glucosyltransferase (P. hybrida,
Q9SBQ2), PL: putative glucosyltransferase (P. lunatus, Q8S3B5), SB:
UDP-glucose: flavonoid 7-O-glucosyltransferase (S. baicalensis,
Q9SXF2), SolB: UDPG glucosyltransferase (S. berthaultii, O24341),
SOB: UDP-glucose glucosyltransferase (S. bicolour, Q9SBL1), ST:
UDP-glucose glucosyltransferase (S. tuberosum, P93789), VV: UDP
flavonoid 3-O-glucosyltransferase (V. vinifera, O22304), ZM: flavonol
3-O-glucosyltransferase (Z. mays, P16166).
Fig. 4. Purity of arbutin synthase wild-type and mutant enzymes after
Ni
2
-nitrilotriacetic acid chromatography SDS/PAGE and staining by
Coomassie-blue. (I, marker proteins; II, AS-WT; III, AS-L86I; IV,
AS-A204V; V, AS-E368D; VI, AS-E368A; VII, AS-H360K; VIII,
AS-H360R; IX, H360E; w, arbutin synthase and its muteins).
Table 1. Comparison of kinetic parameters of wild-type and muteins of arbutin synthase-(His)
6
expressed in E. c oli. Values of K
m
and k
cat
were
calculated from Lineweaver–Burk plots using vanillin as substrate (n.d. ¼ not detectable, detection limit < 10 pkatÆmg
)1
).
Enzyme
arbutin synthase
K
m
vanillin
[lmolÆL
)1
]
V
max
vanillin
[pkat]
Specific activity hydroquinone
[nkatÆmg
)1
]
Specific activity vanillin
[nkatÆmg
)1
]
k
cat
vanillin
[s
)1
]
k
cat
/K
m
vanillin
[LÆmol
)1
Æs
)1
]
Wild type 440 46.5 202.9 24.1 2.59 5886.4
L86I 452 38.3 180.4 20.4 1.84 4070.8
A204V 706 12.3 139.1 13.1 1.30 1841.4
E368D 411 1.9 17.8 0.9 0.12 292.0
E368A 398 6.2 24.0 1.1 0.08 201.0
H360R – – 0.22 n.d. – –
H360K – – 0.17 n.d. – –
H360E – – 0.05 n.d. – –
536 T. Hefnerand J. Sto
¨
ckigt (Eur. J. Biochem. 270) Ó FEBS 2003
the nucleophile. We were able to prove for the first time the
suggested model bysite-directedmutagenesisof arbutin
synthase. First the E368D mutein was generated. Deter-
mination of the kinetic properties of this mutein indeed
indicated a clear decrease of activity, e.g. of the specific
enzyme activity more than 10- and 25-fold for hydroqui-
none and vanillin, respectively. Also the k
cat
/K
m
value for
the substrate vanillin decreased dramatically in comparison
withthewild-type(Table1).InthecasethattheGlu(orthe
Asp) residue at this position is really crucial for the sugar
transfer as a nucleophile, its exchange against another
amino acid, e.g. by a neutral one, must lead to a total loss of
enzyme activity as it has been discussed [8]. As a
consequence we created and tested the mutant arbutin
synthase E368A, in which the putative Glu was exchanged
by Ala. But this mutein still exhibited remarkable enzyme
activity (Table 1). Each of the measured kinetic parameters
of this mutant enzyme with regard to vanillin were in the
same range as those obtained for the former mutant enzyme
E368D. The specific activities of mutein E368A were even
slightly higher with both substrates hydroquinone and
vanillin than those of E368D.
This mutagenesis experiment obviously excludes Glu368
as the nucleophile. A nucleophilic residue is, however, a
prerequisite for the S
N
2 reaction, leading from the a-con-
figured UDP-glucose to the b-configured glucosylated
product. As shown by our alignment study Glu384 might
remain as an appropriate candidate (Fig. 3). But Glu384 (in
AS) is far from Glu368 and appears not to be strictly
conserved. Whereas it is detected in five of the 24 sequences
it is replaced by its homologue Asp in the remaining 19
enzymes. Future experiments must show, whether the both
residues at position 384 provide the nucleophile for the
reaction instead of Glu368 or whether an acidic amino acid
outside the PSPG-box may occupy the nucleophilic role.
For deeper investigation of the properties of the mutein
AS-E368A, an additional eight substrates were tested
(Fig. 5). By determination of the specific activities towards
these substrates, it was possible to compare these with the
activities obtained with the AS wild-type enzyme
(Table 2). Surprisingly changes of the relative enzyme
activities were not at the same level. Eugenol, for instance,
was not glucosylated at all. If substitution of Glu by Ala
causes an affect at the acidic catalytic centre only,
activities towards different substrates should not change.
By the obtained results we may conclude, that replacing
Glu by Ala does not only affect the NRD, but also has
an influence on recognition of the substrates. It may be
that the observed effect is due to alteration of the
stereochemical and electronic situation at the substrate
binding pocket, but at the present time these domains of
glucosyltransferases are also unknown. Therefore any
conclusions drawn on correlations between enzyme acti-
vity towards different substrates and mutations must be
considered tentative until confirmed by X-ray crystallo-
graphic analyzes.
Mutations at the second typical and conserved residue in
position 1, which is His360 in arbutin synthase, did not
support at all the suggested reaction mechanism model.
For this model it has been assumed that mutation of His
by other basic amino acids such as Arg or Lys would
probably be tolerated by enzymes of the NRD1 family. In
contrast to this theory, the appropriate mutant enzymes of
arbutin synthase, H360R and H360K, showed such small
conversion rates that determinations of K
m
, V
max
or k
cat
values were not attainable and only measurement of the
Table 2. Specific activities of AS-WT and E368A. For better comparison, the relative activities of the wild-type enzyme were divided through the
activity values obtained by the mutein E368A.
Substrate
AS-WT specific activity
[nkatÆmg
)1
]
relative activity
[%]
AS-E368A specific activity
[nkatÆmg
)1
]
relative activity
[%]
relative activity WT/
relative activity E368A
Hydroquinone 208.9 100 28.9 100 1.0
b-Naphthol 38.4 18.4 2.1 7.3 2.5
8-Hydroxyquinoline 24.9 11.9 1.0 3.5 3.4
3-Methoxyphenol 24.7 11.8 0.6 2.1 5.6
Vanillin 22.2 10.6 1.8 6.2 1.7
Resorcinol 16.3 7.8 0.8 2.8 2.8
4-Hydroxybenzaldehyde 15.3 7.3 0.8 2.8 2.6
Umbelliferone 10.8 5.1 0.9 3.1 1.6
Eugenol 8.5 4.1 < 0.03 < 0.1 > 40
Scopoletin 7.4 3.5 0.6 2.1 1.7
Fig. 5. Structures of substrates that were tested with arbutin synthase
wild-type and E368A mutein.
Ó FEBS 2003 Probingcatalyticdomainsof NRD glycosyltransferases (Eur. J. Biochem. 270) 537
specific activities of both mutants with the natural highly
accepted substrate hydroquinone (K
m
<1l
M
) was pos-
sible. However, the measured enzyme activities were
approximately 1000-fold diminished compared to the
wild-type or even 100-fold smaller as determined for
the above discussed mutein E368A. We therefore believe,
that the functional role of His in position 1 of NRD1b
family members is apparently more crucial than has
previously been accepted. This observation is additionally
supported by a further mutation experiment. If glycosyl-
transferases, which catalyze sugar transfer with retention of
configuration, also depend on the presence of a glutamic
acid residue in the same position as histidine occupies in
inverting transferases (position 360), it would be an exciting
challenge to convert an inverting to a retaining enzyme just
by such a point mutation. For that reason we created the
H360E mutant enzyme of arbutin synthase which however,
exhibited an 4000-fold decreased enzyme activity. This
enzyme did in fact, reveal the lowest specific activity of all
the mutant enzymes described here. Nevertheless, arbutin
could be enzymatically synthesized with high amounts of
this mutein (approximately 50-fold compared to the
standard assay) and much longer incubation times
(approximately 200-fold) because of excellent enzyme
expression and simple purification. The isolated and
purified glucosidic product was clearly identified as the
O-b-glucoside of hydroquinone, because it resisted incuba-
tion in the presence of a-glucosidase. In contrast, it was
completely hydrolyzed in the presence of b-glucosidase as
shown by TLC and HPLC analysis. This experiment is
again not in agreement with the suggested mechanism for
the glycosyltransferase reaction which, would lead to the
hydroquinone-O-a)glucoside and not to the O-b-glucoside
arbutin as observed.
Conclusions
As arbutin synthase fulfils all the requirements of a member
of the NRD1b enzyme family, the recent suggestion on
catalytic important amino acids of glycosyltransferases,
which is based on sequence alignment studies, is not
satisfactory due to the results of the site-directed mutagen-
esis experiments presented here. The question concerning
the mechanism of one of the basic reactions in cells, the
transfer of a sugar moiety during the formation of an a-or
b-glucoside, still awaits an answer. Especially when consid-
ering the results obtained by the mutein AS-E368A, where
obviously the substrate recognition site was affected by this
point-mutation. Crystallization and cocrystallization of
such a glucosyltransferase with nucleotide sugars and
substrates followed by X-ray analysis might be the best
strategy for future success in elucidating the molecular
nature of the glucosylation process.
Acknowledgements
The financial support provided by Deutsche Forschungsgemeinschaft
(Bonn, Germany) andby the Fonds der Chemischen Industrie
(Frankfurt/Main, Germany) is highly appreciated. We also thank
J. Arend (Mainz) for advice in enzyme purification and W. E. Court
(Mold) for linguistic help.
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538 T. Hefnerand J. Sto
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. Probing suggested catalytic domains of glycosyltransferases
by site-directed mutagenesis
Tobias Hefner and Joachim Sto¨ ckigt
Lehrstuhl fu
¨
r Pharmazeutische. a member of the NRD1b
family of glycosyltransferases. This enzyme was used to
prove, by site-directed mutagenesis, suggested catalytic
domains and reaction