Probing suggested catalytic domains of glycosyltransferasesby site-directed mutagenesis Tobias Hefner and Joachim Sto¨ckigt Lehrstuhl fu¨r Pharmazeutische Biologie, Johannes Gutenberg-Un
Trang 1Probing suggested catalytic domains of glycosyltransferases
by site-directed mutagenesis
Tobias Hefner and Joachim 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, by site-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 mutagenesis and 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 reverse genetics allowed us to use the enzyme as one of the most 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 QuickChangeTMSite-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 was submitted to GenBank with accession number
AJ310148.
Note: Dedicated to Professor Zenk on his seventieth birthday.
(Received 15 April 2002, revised 27 September 2002,
accepted 2 December 2002)
Trang 2CCCG-3¢, L(86)Irev: 5¢-CGGGAGAGAGCGAGTGAT
GGTGATACAAATACGGGTC-3¢; A(204)Vfor: 5¢-GGC
CAAGAGATACCGGTTAGTTGAGGGTATCATGG-3¢, A(204)Vrev: 5¢-CCATGATACCCTCAACTAACCGG
ATTCTTGACAGTGTAGTTAATGGGGTGCCG-3¢;
ATTCTTGCGAGTGTAGTTAATGGGGTGCCG-3¢;
E(368)Arev: 5¢-CGGCACCCCATTAACTACACTCGCA
AGAATAGAGTTCC-3¢; H(360)Rfor: 5¢-GGGTGGATT
TCTAACCCGATGCGGGTGGAAC-3¢; H(360)Rrev:
5¢-GTTCCACCCGCATCGGGTTAGAAATCCACCC-3¢;
H(360)Kfor: 5¢-CGGGTGGATTTCTAACCAAGTGCG
CTTGGTTAGAAATCCACCC-3¢; H(360)Efor: 5¢-CG
GGTGGATTTCTAACCGAGTGCGGGTGGAAC-3¢;
H(360)Erev: 5¢-GTTCCACCCGCACTCGGTTAGAAA
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)1ampicillin
and 25 mgÆL)1 kanamycin, were grown overnight at
200 r.p.m and 37C These cultures which were used to
inoculate 2 L LB medium (antibiotics as before plus 0.3 mM
IPTG), were cultivated at 100 r.p.m and 25C After 24 h
the cells were harvested by centrifugation at 4000 g for
10 min The resulting pellets were resuspended in 50 mL
buffer (K2HPO4 50 mM, pH 8.0, 300 mM NaCl, 10 mM
imidazole and 20 mMb-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 mM imidazole the enzyme was eluted by a
linear gradient (20–250 mM 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 mM hydroquinone or the substrates tested (Fig 5),
2 mM UDP-Glu, 100 mM 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 50C 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 (H3PO4) 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 lL water, containing 100 mMTris, pH 7.5 and 20 mM mercaptoeth-anol was prepared After incubation at 37C for 17 h, the reaction was terminated with 300 lL MeOH, centrifuged and freeze-dried The residue was dissolved in 500 lL MeOH/H2O (7 : 3) and applied to a TLC plate (Silica gel 60
F254, solvent system EtOAc/MeOH/H2O (7 : 2 : 1)) The bands identified as the glucoconjugates were scratched out and eluted with 1.5 mL CH2Cl2/MeOH (7 : 3) Ten micro-litres of this fraction were mixed with 190 lL MeOH and 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 lL H2O To
25 lL of this solution 175 lL citrate buffer (100 mM,
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 mMK2HPO4(pH 6.0) were added After incubation at
37C 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 expressed in an active form in E 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 serpentina or heterologously expressed
in E coli.
Trang 3at 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 catalytic domains of 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 NRD1bS and large 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-directed mutagenesis 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 Ni2+-nitrilotriacetic acid columns Based on Coomassie-blue staining all these mutant enzymes showed high purity (Fig 4) Because the
Kmvalue of the natural substrate of the synthase, hydro-quinone, is extremely small (< 1 lM) and very difficult to measure, we used the substrate vanillin for the determin-ation of kinetic parameters and enzyme activity (Km, Vmax,
kcat, kcat/Km) 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 Km-value and approximately 18% decrease of Vmax Replacement of Ala204 to Val caused a greater decrease of the catalytic efficiency (kcat/Km), 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).
Trang 4mutant 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 of catalytic 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; :, 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
Ni2-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 coli 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)1Æs)1]
Trang 5the nucleophile We were able to prove for the first time the
suggested model by site-directed mutagenesis of 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 kcat/Kmvalue for
the substrate vanillin decreased dramatically in comparison
with the wild-type (Table 1) In the case that the Glu (or the
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 SN2 reaction, leading from the
a-con-figured UDP-glucose to the b-cona-con-figured 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 Km, Vmaxor kcat 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
Fig 5 Structures of substrates that were tested with arbutin synthase wild-type and E368A mutein.
Trang 6specific activities of both mutants with the natural highly
accepted substrate hydroquinone (Km< 1 lM) 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) and by 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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