Aspergillus nidulans α-galactosidase of glycoside hydrolase family 36 catalyses the formation of α-galacto-oligosaccharides by transglycosylation

Article abstract of DOI:10.1111/j.1742-4658.2010.07763.x

The α-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic α-galactosidases and -galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L^-1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with α-(1→6) regioselectivity from 40 mm 4-nitrophenol -d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol α-d-Galp-(1→6) α-d-Galp, α-d-Galp- (1→6) α- d-Galp-(1→6) α-d-Glcp and α-d-Galp- (1→6) α- d-Galp-(1→6) α-d-Glcp-(1→2) α-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol -d-galactopyranoside (40 mm), -(1→6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 -galactosidase as the template and superimposition of melibiose from the complex with human GH27 α-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred -galactosyl to 6-OH of the terminal residue in the -linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the -linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. α-d-Galp-(1→6) α-d-Manp; α-d-Galp-(1→6) α- d-Glcp-(1→4) α-d-Glcp; α-d-Galp-(1→6) α- d-Galp-(1→4) α-d-Fruf; α-d-Galp-(1→6) α-d-Glcp-(1→1) α-d-Glcp; and α-d-Galp-(1→6) α- d-Glcp-(1→3) α-d-Fruf.

Full text of DOI:10.1111/j.1742-4658.2010.07763.x

Aspergillus nidulans a-galactosidase of glycoside
hydrolase family 36 catalyses the formation of
a-galacto-oligosaccharides by transglycosylation
1
1
2
3
1
Hiroyuki Nakai , Martin J. Baumann , Bent O. Petersen , Yvonne Westphal , Maher Abou Hachem ,
1
2
3
1
Adiphol Dilokpimol , Jens Ø. Duus , Henk A. Schols and Birte Svensson
1
2
3
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark
Carlsberg Laboratory, Valby, Denmark
Laboratory of Food Chemistry, Wageningen University, The Netherlands
Keywords
The a-galactosidase from Aspergillus nidulans (AglC) belongs to a phyloge-
netic cluster containing eukaryotic a-galactosidases and a-galacto-oligosac-
charide synthases of glycoside hydrolase family 36 (GH36). The
acceptor specificity; carbohydrate structural
analysis; transglycosylation; a-galacto-
oligosaccharides; a-galactosidase
)
1
recombinant AglC, produced in high yield (0.65 gÆL culture) as His-tag
fusion in Escherichia coli, catalysed efficient transglycosylation with
a-(1 fi 6) regioselectivity from 40 mm 4-nitrophenol a-d-galactopyranoside,
melibiose or raffinose, resulting in a 37–74% yield of 4-nitrophenol
a-d-Galp-(1 fi 6)-d-Galp, a-d-Galp-(1 fi 6)-a-d-Galp-(1 fi 6)-d-Glcp and
a-d-Galp-(1 fi 6)-a-d-Galp-(1 fi 6)-d-Glcp-(a1 fi b2)-d-Fruf (stachyose),
respectively. Furthermore, among 10 monosaccharide acceptor candidates
Correspondence
B. Svensson, Enzyme and Protein
Chemistry, Department of Systems Biology,
Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby,
Denmark
Fax: +45 4588 6307
(
400 mm) and the donor 4-nitrophenol a-d-galactopyranoside (40 mm),
Tel: +45 4525 2740
a-(1 fi 6) linked galactodisaccharides were also obtained with galactose, glu-
cose and mannose in high yields of 39–58%. AglC did not transglycosylate
monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabi-
nose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose
and l-rhamnose. Structural modelling using Thermotoga maritima GH36
a-galactosidase as the template and superimposition of melibiose from the
complex with human GH27 a-galactosidase supported that recognition at
subsite +1 in AglC presumably requires a hydrogen bond between 3-OH
and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl
group. In addition, successful transglycosylation of eight of 10 disaccharides
E-mail: bis@bio.dtu.dk
(
Received 17 May 2010, revised 2 July
010, accepted 5 July 2010)
2
doi:10.1111/j.1742-4658.2010.07763.x
(
400 mm), except xylobiose and arabinobiose, indicated broad specificity for
interaction with the +2 subsite. AglC thus transferred a-galactosyl to 6-OH
of the terminal residue in the a-linked melibiose, maltose, trehalose, sucrose
and turanose in 6–46% yield and the b-linked lactose, lactulose and cello-
biose in 28–38% yield. The product structures were identified using
NMR and ESI-MS and five of the 13 identified products were novel,
i.e. a-d-Galp-(1 fi 6)-d-Manp; a-d-Galp-(1 fi 6)-b-d-Glcp-(1 fi 4)-d-Glcp;
a-d-Galp-(1 fi 6)-b-d-Galp-(1 fi 4)-d-Fruf; a-d-Galp-(1 fi 6)-d-Glcp-(a1 fi a1)-
d-Glcp; and a-d-Galp-(1 fi 6)-a-d-Glcp-(1 fi 3)-d-Fruf.
Abbreviations
AglC, a-galactosidase from Aspergillus nidulans; GalA, a-galactosidase from Thermotoga maritima; GH, glycoside hydrolase family;
HPAEC-PAD, high-performance anion-exchange chromatography equipped with pulsed amperometric detection; pNP, 4-nitrophenol;
pNPaAra, 4-nitrophenyl a-L-arabinopyranoside; pNPaAraf, 4-nitrophenyl a-L-arabinofuranoside; pNPaGal, 4-nitrophenyl a-D-galactopyranoside;
pNPaGalNAc, 4-nitrophenyl N-acetyl a-D-galactosaminiside; pNPaGlc, 4-nitrophenyl a-D-glucopyranoside; pNPaGlcNAc, 4-nitrophenyl N-acetyl
a-D-glucosaminiside; pNPaMan, 4-nitrophenyl a-D-mannopyranoside; pNPaRha, 4-nitrophenyl a-L-rhamnopyranoside; pNPaXyl, 4-nitrophenyl
a-D-xylopyranoside; pNPbGal, 4-nitrophenyl b-D-galactopyranoside.
3
538
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS

H. Nakai et al.
Transglycosylation by A. nidulans a-galactosidase
Introduction
a-Galactosidases (EC 3.2.1.22) are exo-acting glycoside
hydrolases that catalyse the release of galactose from
a-galacto-oligosaccharides, e.g. melibiose [a-d-Galp-(1 fi
maltose as acceptors, respectively [30,31]. Detailed
acceptor specificity, however, has not been investigated
previously for GH36 a-galactosidases and a survey is
presented here of suitable acceptors. The results
obtained may also provide a certain insight into speci-
ficity in hydrolysis catalysed by GH36.
6
)-d-Glcp], raffinose [a-d-Galp-(1 fi 6)-d-Glcp-(a1 fi
b2)-d-Fruf] and stachyose [a-d-Galp-(1 fi 6)-a-d-Galp-
1 fi 6)-d-Glcp-(a1 fi b2)-d-Fruf], polymeric galacto-
(
mannans containing a-(1 fi 6) linked galactosyl resi-
dues bound to a b-(1 fi 4) mannan backbone and
galactolipids [1]. a-Galactosidases occur widely in bac-
teria [2–11], fungi [12–15], plants [16,17] and animals
Certain
a-galacto-oligosaccharides
have
been
reported to be candidates for health-promoting prebi-
otic food ingredients [36–38] because a-galactosidase
is lacking in the human gastrointestinal tract and
a-galacto-oligosaccharides can be digested by the intes-
tinal microbiota and stimulate growth of beneficial
Bifidobacteria and Lactobacilli. In the present study,
efficient transglycosylation catalysed by Aspergillus
nidulans FGSC GH36 a-galactosidase (AglC), which
was produced recombinantly in Escherichia coli,
resulted in chemo-enzymatic synthesis of 13 a-galacto-
oligosaccharides, including five not reported previ-
ously, representing novel prebiotics oligosaccharide
candidates. The enzymatic properties of AglC are
described with focus on specificity and regioselectivity
in transglycosylation using 4-nitrophenol a-d-galacto-
pyranoside (pNPaGal) as the donor and different
mono- and disaccharides as acceptors. Furthermore,
structural modelling of AglC using the three-dimen-
sional structure of GalA [24] as the template and
superimposition of an equilibrium mixture of a- and
b-galactose from Oryza sativa a-galactosidase [28],
N-acetyl-a-galactosamine from Gallus gallus a-N-acet-
ylgalactosaminidase [29] and melibiose from human
a-galactosidase [27] complexes of GH27 were per-
formed to illustrate the donor and acceptor specificity
and the regioselectivity of AglC.
[
18,19] and have been classified based on substrate
specificity [20] and sequence similarity [21]. With
regard to substrate specificity, one type of a-galactosi-
dase from fungi and plants acts specifically on a-galac-
to-oligosaccharides, whereas another type is able to
degrade both these and polymeric galactomannans.
a-Galactosidases are classified into glycoside hydrolase
families GH4, GH27, GH36, GH57, GH97 and
GH110 [21]. Eukaryotic a-galactosidases belong to
GH27 and GH36, which form clan GH-D together
with GH31 (http://www.cazy.org/) [21] and are consid-
ered to have a common evolutionary origin [22].
Hydrolysis of GH27 [23] and GH36 [24] catalysed
by a-galactosidases proceeds via a double-displacement
mechanism, resulting in net retention of the stereo-
chemistry at the anomeric centre [25]. First, the general
acid catalyst protonates the glycosidic oxygen concom-
itantly with bond cleavage and the catalytic nucleo-
phile forms a covalent glycosyl-enzyme intermediate by
direct attack at the anomeric centre. In the next step,
water is deprotonated by the general base catalyst and
attacks the anomeric centre, releasing the carbohydrate
moiety. For GH36, the nucleophile and the acid ⁄base
catalysts of Thermotoga maritima a-galactosidase
(
GalA) were identified by mutational and structural
Results and Discussion
analyses to be Asp327 and Asp387, respectively [24].
In GH27, both labelling with mechanism-based inhibi-
tors [26,27] and crystal structures of ligand complexes
of a-galactosidase [27,28] and a-N-acetylgalactosamini-
dase (EC 3.2.1.49) [29] identified the catalytic residues,
whereas no crystal structure was available of a ligand
complex for GH36.
Sequence similarity of AglC
The amino acid sequence of AglC, deduced from aglC
(GenBank, gi: 40739585), shows 6–79% sequence iden-
tity and 17–90% sequence similarity with different
functionally characterized GH36 members (Table 1).
Phylogenetically, AglC occurs in the cluster of eukary-
otic a-galactosidases (Fig. 1) and has highest identity
(79%) to a-galactosidase of Aspergillus niger [12],
whereas it has low identity (6–10%) to plant alkaline
a-galactosidases involved in the metabolism of raffi-
nose, stachyose and polymeric galactomannans, serving
as storage carbohydrates in many botanical families
[16,17], and to raffinose (EC 2.4.1.82) [39] and stachy-
ose synthases (EC 2.4.1.67) [40,41], which catalyse the
a-Galactosidases have been reported to form a-ga-
lacto-oligosaccharides at high substrate concentrations
by catalysing the transfer of a galactosyl moiety to an
acceptor with a-(1 fi 3), a-(1 fi 4) or a-(1 fi 6) regi-
oselectivity [2–4,30–35]. Chemo-enzymatic synthesis
using suitable donor and acceptor pairs can produce
raffinose, a-d-Galp-(1 fi 6)-b-d-Galp-(1 fi 4)-d-Glcp
and a-d-Galp-(1 fi 6)-a-d-Glcp-(1 fi 4)-d-Glcp with
melibiose as the donor and sucrose, lactose and
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS
3539

Transglycosylation by A. nidulans a-galactosidase
H. Nakai et al.
Table 1. Amino acid sequence comparison of AglC from Aspergillus nidulans FGSC with functionally characterized GH36 enzymes. Similari-
ties of amino acid sequences were determined using the BLASTP program (Swiss-Prot ⁄ TrEMBL database).
Swiss-Prot ⁄ TrEMBL
accession no.
Identity (%)
Similarity (%)
Gap (%)
a-Galactosidase
Aspergillus niger CBS 120.49 ⁄ N400
Penicillium sp. F63 CGMCC1669
Geobacillus stearothermophillus NUB3621
Clostridium stercorarium F-9
Lactobacillus plantarum ATCC 8014
Carnobacterium piscicola strain BA
Lactococcus raffinolactis ATCC 43920
Mycocladus corymbiferus IFO 8084
Lactobacillus fermentum CRL722
Bifidobacterium bifidum NCIMB 41171
Bifidobacterium breve 203
Q9UUZ4
A4Z4V0
Q9X624
Q84IQ0
Q9L905
Q93DW8
Q7XIP3
Q9P8N4
Q6IYF5
79
68
39
36
33
34
36
28
27
23
21
10
8
90
81
54
54
51
53
54
44
45
37
36
19
18
0
2
6
7
5
6
6
11
8
Q1KTD9
Q2XQ11
Q9WXC1
O33835
9
12
38
32
Thermus sp. strain T2
Thermotoga maritima MSB8
a-N-Acetylgalactosaminidase
Clostridium perfringens
Q8XNK8
8
8
20
20
25
25
Raffinose synthase
Glycine max CV. CLARK63
BD953761
Stachyose synthase
Pisum sativum L cv. Wunder von Kelevedon
Vigna angularis Ohwi et Ohashi
Q93XK2
Q9SBZ0
6
6
17
17
20
21
formation of storage oligosaccharides by transglycosy-
lation using galactinol [O-a-d-Galp-(1 fi 1)-l-myo-ino-
sitol] as the donor. This relationship motivated the
detailed analysis of the capacity and specificity of AglC
in transglycosylation reactions.
binant AglC was estimated to be 335 kDa by gel filtra-
tion chromatography, indicating that AglC is
a
tetramer in solution, similar to several bacterial and
fungal GH36 a-galactosidases [2,5,6,8,10,14,15].
Other bacterial and fungal GH36 a-galactosidases
were found to be dimers [4], trimers [3] or octamers
[
11], whereas plant alkaline a-galactosidases [16] and
Overproduction in E. coli and purification of AglC
bacterial a-N-acetylgalactosaminidases [46,47] were
monomers.
Similar to other fungal GH36 a-galactosidases [13–15],
AglC has a signal peptide (Met1-Ala26; predicted by
wolf psort [42] and signalp [43]), and the bioinfor-
matic analysis suggests that AglC is an extracellular
GH36 a-galactosidase. Previously, heterologous expres-
sion in Pichia pastoris X-33 and partial characteriza-
tion was described for AglC, together with a large
number of cell wall polysaccharide-degrading enzymes
annotated in the A. nidulans genome [12,44]. However,
because this recombinant AglC was produced with sig-
nal peptide, we overproduced AglC in E. coli by
expression of aglC encoding mature protein, isolated
from genomic DNA of the P. pastoris transformant
Enzymatic properties of AglC
AglC hydrolysed pNPaGal, but not the pNP glycosides
of N-acetyl a-d-galactosamine (pNPaGalNAc, i.e.
the substrate for GH36 a-N-acetylgalactosaminidase
[45,47]), b-d-galactopyranose (pNPbGal), a-d-gluco-
pyranose (pNPaGlc), N-acetyl a-d-glucosamine (pNPa
GlcNAc), a-d-xylopyranose (pNPaXyl), a-d-manno-
pyranose (pNPaMan), a-l-arabinopyranose (pNPaAra),
a-l-arabinofuranose (pNPaAraf) and a-l-rhamnopyra-
nose (pNPaRha) (less than 10 lm pNP liberated in the
reaction mixture). AglC is thus an a-galactosidase, as
also suggested by the sequence similarity (Table 1,
Fig. 1). The pH optimum of AglC catalysed hydrolysis
of pNPaGal was 5.0 (Fig. 2A) as found for bacterial
[4,7,9,34] and other fungal a-galactosidases [13–15],
whereas plant alkaline a-galactosidases have pH
optima of 7.5–8.5 [16,17]. AglC showed good stability
(
see Experimental procedures) under strict control of
the cold shock promoter cpsA and the lac operator
45]. The resulting AglC His-tag fusion was purified by
[
nickel chelating chromatography in a yield of 1.3 g
from 2 L culture and migrated in SDS ⁄ PAGE as a sin-
gle band with an estimated molecular mass of 83 kDa
(Fig. S1). Moreover, the molecular mass of the recom-
3
540
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS

H. Nakai et al.
Transglycosylation by A. nidulans a-galactosidase
Fig. 1. Phylogenetic tree constructed based on deduced full-length amino acid sequences of functionally characterized GH36 glycosidases
and synthases using CLUSTALW. The rectangular cladogram tree was generated with TREEVIEW version 1.6.6 software. Values at nodes repre-
sent the percentage of bootstrap confidence level on 1000 resamplings. Bacterial a-galactosidases: Bifidobacterium adolescentis DSM
2
0083 Aga (GenBank, gi: 14495552), Bifidobacterium bifidum NCIMB 41171 MelA (gi: 90655076), Bifidobacterium breve 203 Aga2
gi:82468523), Clostridium stercorarium F-9 Aga36A (gi: 28268728), Escherichia coli K-12 RafA (gi: 147505), Geobacillus stearothermophilus
KVE39 AgaA (gi: 12331004), Geobacillus stearothermophilus NUB3621 AgaN (gi: 4567098), Lactococcus raffinolactis ATCC 43920 AgA (gi:
2450781), Lactobacillus fermentum CRL722 MelA (gi: 47717086), Lactobacillus plantarum ATCC8014 MelA (gi: 15042935), Streptococ-
cus mutans strain Ingribitt Aga (gi:153736), Thermotoga maritima MSB8 GalA (gi: 55165925), Thermotoga neapolitana 5068 AglA (gi:
237318), Thermus brockianus ITI360 AgaT (gi: 4928639), Thermus sp. strain T2 AglA (gi: 4587043) and Thermus thermophilus TH125 AgaT
gi: 4894857); bacteria a-N-acetylgalactosaminidase: Clostridium perfringens ATCC 10543 AagA (gi: 22651784); fungal a-galactosidases:
(
3
3
(
Aspergillus nidulans FGSC A4 AglC (gi: 40739585), Aspergillus niger CBS 120.49 ⁄ N400 AglC (gi: 6624914) and Penicillium sp. F63
CGMCC1669 Agl1 (gi: 85375918); plant alkaline a-galactosidase: Cucumis melo L. Aga1 and Aga2 (gi: 29838629 and gi: 29838631),
Pisum sativum L. cv. Kelvedon Wonder AGa1 (gi: 148925503), Tetragonia tetragonioides TtAga1 (gi: 209171772) and Zea mays L. ZmAGA1
and ZmAGA3 (gi: 68270843 and gi: 33323027); raffinose synthase: Glycine max CV. CLARK63 Ras (gi: 67587384); stachyose synthases:
Hordeum vulgare subsp. vulgare Sip1 (gi: 167100), Pisum sativum L. cv. Wunder von Kelvedon Sts1 (gi: 13992585), Stachys sieboldii STS
(gi: 19571727) and Vigna angularis Ohwi wt Ohashi VaSTS1 (gi: 6634701).
at pH 3.6–9.9 (Fig. 2B), maximum activity at 50 ꢀC
Fig. 2C) and retained > 95% activity after 15 min
similar to bacterial a-galactosidases [5,6,10,20],
whereas plant alkaline a-galactosidase showed 18-fold
higher kcat ⁄ Km for raffinose than melibiose [16].
(
incubation up to 45 ꢀC at pH 5.0 (Fig. 2D).
AglC hydrolysed the a-galactosidic linkage in
pNPaGal, melibiose [a-d-Galp-(1 fi 6)-d-Glcp] and
raffinose [a-d-Galp-(1 fi 6)-d-Glcp-(a1 fi b2)-d-Fruf],
but failed to cleave off a-(1 fi 6) galactosyl branches
in galactomannan [48]. AglC thus belongs to the cate-
gory of exo-acting fungal and plant a-galactosidases
hydrolysing a-galacto-oligosaccharides [20]. The cata-
Transglycosylation and acceptor specificity of
AglC
Phylogenetically AglC is in a cluster including eukary-
otic a-galactosidases (Fig. 1) and also has sequence
similarity (17–20%) to raffinose and stachyose synthas-
es (Table 1). These enzymes synthesize raffinose and
stachyose by transglycosylation, and it is shown here
that AglC also efficiently catalysed transglycosylation
of pNPaGal as monitored by TLC (Fig. 3A) and
HPLC (Fig. 3D). The product was obtained in 74%
yield after 1 h and ESI-MS showed m⁄ z of 486 corre-
lytic efficiency (kcat ⁄ K ) towards pNPaGal was two
m
orders of magnitude higher than for these oligosaccha-
rides due to the lower Km value (Table 2). This repre-
sents the first kinetic analysis of a fungal GH36
a-galactosidase and AglC gave approximately two-fold
lower kcat ⁄ Km for raffinose compared with melibiose,
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS
3541

Transglycosylation by A. nidulans a-galactosidase
H. Nakai et al.
A
B
C
D
Fig. 2. Effect of pH and temperature on the
activity and stability of AglC. (A) pH depen-
dence for hydrolysis of pNPaGal by 0.27 nM
AglC (
pH 2.3–11.9. (B) pH stability of 1.6 nM AglC
s) in 90 mM Britton-Robinson buffer pH
.3–11.9. (C) Temperature activity depen-
dence for 4.1 nM AglC ( ) at 20–90 ꢀC with
0 min reaction. (D) Stability of 9.0 nM AglC
h) in the temperature range 20–90 ꢀC for
5 min. Each experiment was carried out in
•
) in 40 mM Britton-Robinson buffer
(
2
1
(
1
triplicate. Standard deviations are shown as
error bars.
+
+
Table 2. Kinetic parameters for hydrolysis of pNPaGal, melibiose
and raffinose by AglC. Parameters are calculated from the initial
velocities of release of pNP from pNPaGal and of galactose from
melibiose and raffinose at different substrate concentrations (see
Experimental procedures).
Na ) and galactosyl-raffinose (C H O + Na )
24 42 21
and two-dimensional NMR identified the oligosaccha-
rides as a-d-Galp-(1 fi 6)-a-d-Galp-(1 fi 6)-d-Glcp
and a-d-Galp-(1 fi 6)-a-d-Galp-(1 fi 6)-d-Glcp-(a1 fi
b2)-d-Fruf (stachyose), respectively (Table S1). AglC
thus catalysed efficient transglycosylation with a-1,6-
regioselectivity at a lower concentration (40 mm) of
melibiose and raffinose compared with a-galactosidases
of Bifidobacterium [3,4,32] and Lactobacillus [34] of the
prokaryotic cluster (Fig. 1) at a higher concentration
of melibiose (0.1–1.2 m) and raffinose (0.46 m) result-
ing in 11–33% and 26% yield, respectively.
)
1
)
)1
(s)1ÆmM)1
)
Substrate
K
m
(mM)
k
cat (s
k
catÆK
m
pNPaGal
Melibiose
Raffinose
0.27 ± 0.01
12 ± 0.12
15 ± 0.57
1278 ± 38
1067 ± 26
586 ± 17
4730
88.9
39.1
+
sponding to the calculated value of the Na adduct of
This important transglycosylation catalysed by AglC
motivated a comprehensive analysis of acceptor speci-
ficity involving 10 mono- and 10 disaccharides
(Table 3) for the formation of a-galacto-oligosaccha-
rides with the donor pNPaGal, which has a good
leaving group. Among the monosaccharides, only
galactose, glucose and mannose were found to be
acceptors, resulting in disaccharide yields of 39–58%
after 3 h reaction (Table 3). Apparently, AglC did not
transfer a-galactosyl to monosaccharides without
6-OH (xylose, l-arabinose, l-fucose and l-rhamnose)
or with axial 3-OH (gulose, allose, altrose and l-rham-
nose), indicating the equatorial 3-OH to be critical for
recognition at subsite +1. On the other hand, analysis
+
1
pNP a-d-galactobioside (C H NO + Na ). H-
1
8
25
13
1
3
and C-NMR spectroscopy indicated the formation of
a
single product, pNP a-d-Galp-(1 fi 6)-d-Galp,
reflecting the regioselectivity of AglC (Table S1). In
contrast, a-galactosidases of Bacillus stearothermophi-
lus and Thermus brockianus were reported to produce
both a-(1 fi 3) and a-(1 fi 6) linked pNP a-d-galacto-
biosides [33]. AglC furthermore synthesized a tri-
(
Fig. 3B, E) and a tetrasaccharide (Fig. 3C, F) from
0 mm melibiose or raffinose in 59 and 37% yields,
4
respectively, during 1 h reaction. ESI-MS showed m ⁄ z
of 527 and 689 corresponding to the calculated values
+
for Na adducts of galactosyl-melibiose (C H O +
32 16
18
3
542
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H. Nakai et al.
Transglycosylation by A. nidulans a-galactosidase
A
B
C
D
E
F
Fig. 3. Monitoring formation of transglycosylation products from pNPaGal (A), melibiose (B) and raffinose (C) by TLC (see Experimental pro-
cedures). Standards: lane S1, pNPaGal and galactose; lane S2, galactose and melibiose; lane S3, glucose; lane S4, galactose and raffinose;
lane S5, sucrose. Products from pNPaGal (D), melibiose (E) or raffinose (F) were reconfirmed by HPLC equipped with a UV or refractive
index (RI) detector and a TSKgel Amide-80 column. The compounds, pNP (1), pNP a-Galp (2), pNP a-Galp-(1 fi 6)-Galp (3), galactose and
glucose (4), melibiose (5), a-Galp-(1 fi 6)-a-Galp-(1 fi 6)-Glcp (6), galactose (7), sucrose (8), raffinose (9) and a-Galp-(1 fi 6)-a-Galp-(1 fi 6)-
Glcp-(a1 fi b2)-Fruf (10) are marked by arrows.
of disaccharide acceptors reflected broad specificity of
subsite +2 and resulted in the formation of eight
trisaccharides, five from a-linked (melibiose, maltose,
trehalose, sucrose, turanose) and three from b-linked
disaccharides (lactose, lactulose, cellobiose) in 26–46%
yield, except for melibiose resulting in only 6% yield
of trisaccharide. Melibiose at a high concentration pos-
sibly competes with the donor pNPaGal having a good
leaving group, resulting in the modest yield. As found
for xylose and l-arabinose, xylobiose and arabinobiose
were not acceptors (Table 3).
rigorous recognition governs the transglycosylation
outcome. Analysis of the product structures (see
below) accordingly indicated strict a-(1 fi 6) regiospec-
ificity for the AglC transglycosylation.
ESI-MS analysis gave m⁄ z signals of 365 or 527
corresponding to calculated molecular masses of Na
+
adducts of mono- or disaccharide acceptor conjugates
of galactose. Chemical shifts for NMR linkage analysis
were assigned based on two-dimensional NMR spectra
(Tables S2 and S3). The formed a-(1 fi 6) linkages
were identified by long-range proton–carbon tree bond
correlation from the nonreducing anomeric proton to
C-6 of the substituted position, as confirmed by inter
NOE correlations. AglC thus recognized the C-6
hydroxymethyl and equatorial 3-OH group of an
aldohexopyranosyl unit at subsite +1 and transferred
a-galactopyranosyl to the 6-OH, resulting in 11
a-(1 fi 6) linked galactosyl-oligosaccharides. These
Progress of transglycosylation during 3 h using
0 mm pNPaGal and 400 mm of the identified 11 func-
4
tional acceptors (see above) (Fig. 4) showed individual
product formation rates and yields, glucose and malt-
ose giving the highest yield, but having the slowest
reaction rate. Noticeably, only one product (Table 3)
was obtained with each acceptor, emphasizing that
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS
3543

Transglycosylation by A. nidulans a-galactosidase
H. Nakai et al.
Table 3. Test of carbohydrate acceptor candidates for transgly-
cosylation as catalysed by AglC using the donor pNPaGal. Products
from 40 mM pNPaGal and 400 mM acceptor were quantified by
HPAEC-PAD using melibiose and raffinose as standards for di- and
trisaccharide products, respectively. Yields are based on the pNPa
Gal concentration (see Experimental procedures).
pyranosyl-(1 fi 3)-d-fructofuranose; a-d-galactopyr-
anosyl-(1 fi 6)-b-d-galactopyranosyl-(1 fi 4)-d-fruc-
tofuranose; and a-d-galactopyranosyl-(1 fi 6)-b-d-
glucopyranosyl-(1 fi 4)-d-glucopyranose.
Substrate recognition by AglC
Yield
Acceptor
Product
(%)
The crystal structure of GalA (PDB ID:1ZY9) [24]
was used as the template to model a truncated AglC
(Gly193–Glu699) comprising b6-b15 of the N-terminal
b-sandwich domain (Gly193–Ala347) and the catalytic
Monosaccharide
Galactose (Gal)
Gulose
a-Galp-(1 fi 6)-Galp
–
39
–
Glucose (Glc)
Allose
a-Galp-(1 fi 6)-Glcp
–
58
–
(
b⁄ a) -barrel (Thr348–Glu699) (Fig. 6). This truncated
8
AglC has 25% sequence identity and 40% sequence
similarity with corresponding GalA domains, whereas
full-length AglC shows only 8% identity and 18% sim-
ilarity. Recognition of a-galactose at subsite )1 of the
modelled AglC structure (Fig. 6A, B) was proposed by
superimposition of a- and b-galactose and N-acetyl-
a-galactosamine, respectively, from complexes with
O. sativa a-galactosidase (1UAS) [28] and G. gallus a-
N-acetylgalactosaminidase (1KTC) [29] both of GH27,
because a ligand complex structure was not available
for GH36. Direct hydrogen bonds appeared in this
model between the a-anomeric 1-OH and the 2-OH of
a-galactose with Asp573, corresponding to Asp387, i.e.
the proposed acid ⁄base catalyst in GalA [24]. Further-
more, Asp511, which corresponds to the predicted cat-
alytic nucleophile Asp327 in GalA [24], presumably
makes a hydrogen bond with O5 of the galactose ring
and direct hydrogen bonds were also suggested
between Lys509 and 3-OH and the axial 4-OH as well
as Asp388 and Asp389 and 4-OH and 6-OH, respec-
tively. These AglC ⁄a-galactose contacts are consistent
with the reported recognition at subsite )1 of the mod-
Mannose (Man)
Altrose
a-Galp-(1 fi 6)-Manp
50
–
–
–
–
–
–
Xylose (Xyl)
L-Arabinose (L-Ara)
L-Fucose (L-Fuc)
L-Rhamnose
Disaccharide
Melibiose
–
–
–
–
a-Galp-(1 fi 6)-a-Galp-(1 fi 6)-Glcp
a-Galp-(1 fi 6)-a-Glcp-(1 fi 4)-Glcp
a-Galp-(1 fi 6)-Glcp-(a1 fi a1)-Glcp
a-Galp-(1 fi 6)-Glcp-(a1 fi b2)-Fruf
a-Galp-(1 fi 6)-a-Glcp-(1 fi 3)-Fruf
–
6
46
26
28
26
–
Maltose
Trehalose
Sucrose
Turanose
Arabinobiose
Lactose
a-Galp-(1 fi 6)-b-Galp-(1 fi 4)-Glcp
a-Galp-(1 fi 6)-b-Galp-(1 fi 4)-Fruf
a-Galp-(1 fi 6)-b-Glcp-(1 fi 4)-Glcp
–
38
38
28
–
Lactulose
Cellobiose
Xylobiose
products include five novel compounds (Fig. 5);
a-d-galactopyranosyl-(1 fi 6)-d-mannopyranose; a-d-
galactopyranosyl-(1 fi 6)-d-glucopyranosyl-(a1 fi a1)-
d-glucopyranose; a-d-galactopyranosyl-(1 fi 6)-a-d-gluco
A
B
C
Fig. 4. Progress of AglC (18 nM) catalysed transglycosylation with different acceptors (400 mM) and pNPaGal (40 mM) as the donor. (A)
Monosaccharides: galactose (•), glucose (s), mannose (h). (B) a-Linked disaccharides: melibiose (•), maltose (s), trehalose (h), sucrose (e),
turanose ( ). (C) b-Linked disaccharides: lactose (D), lactulose ( ) and cellobiose (¤) in 40 mM Na acetate (pH 5.0) at 37 ꢀC for 3 h. Product
concentrations were calculated from peak areas of HPAEC-PAD (linear gradient, 0–75 mM Na acetate in 100 mM NaOH for 35 min; flow rate,
1
0
.35 mLÆmin) ) calibrated with melibiose and raffinose as standards for di- and trisaccharides, respectively.
3
544
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS

H. Nakai et al.
Transglycosylation by A. nidulans a-galactosidase
A
D
E
subsite )1 seems identical to that of the a-galactose
superimposed from the complex with O. sativa a-galac-
tosidase (Fig. 6A,C). Noticeably, direct hydrogen
bonds at subsite +1 are suggested, involving 3-OH
and O5 of the b-glucose moiety in melibose
and Trp358 and Asp511 (the acid ⁄ base catalyst),
respectively. Furthermore, Trp221 from the N-terminal
b-sandwich domain presumably makes a hydrophobic
environment for the C-6 hydroxymethyl group of the
b-glucose moiety. These observations supported that
AglC specifically transferred a-galactopyranosyl to C-6
hydroxymethyl of aldohexopyranosyl units with equa-
torial 3-OH group (Table 3).
B
Conclusion
C
AglC catalysed transglycosylation with remarkable effi-
ciency and selectively transferred a-galactopyranosyl to
the 6-hydroxymethyl group of aldohexopyranoses with
equatorial 3-OH, as indicated by reaction with three
monosaccharide acceptors. AglC also catalysed
transfer to 6-OH of the terminal residue in eight
disaccharides. Five novel a-(1 fi 6) linked galacto-
oligosaccharides were obtained. The very efficient
expression system makes the production of engi-
neered AglC feasible and the transglycosylation has
potential for the design and production of a-galacto-
oligosaccharides with prebiotic effect on human gut
microbiota.
Fig. 5. Structures of novel a-galacto-oligosaccharides produced by
transglycosylation catalysed by AglC. (A) a-Galp-(1 fi 6)-Manp, (B)
a-Galp-(1 fi 6)-Glcp-(a1 fi a1)-Glcp, (C) a-Galp-(1 fi 6)-a-Glcp-(1 fi 3)-Fruf,
(
D) a-Galp-(1 fi 6)-b-Galp-(1 fi 4)-Fruf, (E) a-Galp-(1 fi 6)-b-Glcp-
1 fi 4)-Glcp.
(
elled structure for Bifidobacterium adolescentis GH36
a-galactosidase [35] and AglC specifically hydrolysing
pNPaGal and not pNPaMan and pNPaGlc having
equatorial 4-OH, or pNPaXyl lacking the 6-hydroxym-
ethyl group. Noticeably, the axial 1-OH of a-galactose
projects out of the active site in the model and the cat-
alytic nucleophile Asp511 together with Trp221, from
the N-terminal b-sandwich domain, block for binding
of a b-linked aglycone at subsite +1 in agreement with
AglC not hydrolysing pNPbGal. The bulky Trp221
and Trp570 side chains presumably preclude binding
of the C-2 substituent of N-acetyl-a-galactosamine
Experimental procedures
Materials
Allose, altrose, galactose, glucose, gulose, mannose,
l-fucose, l-arabinose, l-rhamnose, cellobiose, lactose, lactu-
lose, maltose, melibiose, sucrose, trehalose, turanose, raffi-
nose, pNPaAra, pNPaAraf, pNPaGal, pNPaGalNAc,
pNPbGal, pNPaGlc, pNPaGlcNAc, pNPaMan, pNPaRha
and pNPaXyl were purchased from Sigma (St. Louis, MO,
USA). Galactomannans (Carubin type and 98% Guarin
type) and xylose were purchased from Carl Roth (Kar-
lsruhe, Germany). Arabinobiose and xylobiose were from
Megazyme (Bray, Ireland). Other reagents were of analyti-
cal grade and from commercial sources.
(Fig. 6B). The a-N-acetylgalactosaminidase from
G. gallus, in contrast, has a cavity formed by Ser172
and Ala175 in the (b⁄ a) -barrel loop connecting b5
8
and a5 where the N-acetyl group becomes sandwiched
between Tyr176 and Arg197 and Ser172 hydrogen
bonds to the carbonyl oxygen [29].
Sequence analysis
The acceptor recognition at subsite +1 of the mod-
elled AglC structure (Fig. 6C) was further illustrated
by superimposition of melibiose from a complex with
human GH27 a-galactosidase (3HG3) [27]. Proposed
recognition of the a-galactose moiety in melibiose at
clustalw (http://clustalw.ddbj.nig.ac.jp/top-j.html) was
used for the phylogenetic analysis using full-length amino
acid sequences of functionally characterized GH36 members
(http://www.cazy.org/fam/GH36.html). A rectangular clad-
ogram tree was generated using treeview version 1.6.6
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS
3545

Transglycosylation by A. nidulans a-galactosidase
H. Nakai et al.
software and a bootstrap test based on 1000 resampl-
ings (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).
Protein localization and signal peptides were predicted
using wolf psort (http://wolfpsort.org/) [42] and signalp
3.0 server (http://www.cbs.dtu.dk/services/SignalP/) [43],
respectively.
A
B
Fig. 6. Stereo views of presumed ligand
interactions with the active site of AglC.
(
A) An equilibrium mixture of a- and b-galac-
tose (pink) from the complex with O. sativa
a-galactosidase (1UAS) [28] superimposed
on the structure AglC modelled using
T. maritima GH36 a-galactosidase (GalA,
1ZY9) [24] as a template. Suggested hydro-
gen bonds are shown as dotted lines.
Asp511 and Asp573 are predicted to be
nucleophile (nu) and acid ⁄ base (a ⁄ b) cata-
lysts, respectively, by sequence alignment
with GalA [24]. (B) N-acetyl-a-galactosamine
C
(
as a yellow stick) from the complex with
G. gallus a-N-acetylgalactosaminidase (as
yellow lines; 1KTC) [29] superimposed on
the modelled AglC structure (grey lines).
˚
A hydrogen bond (2.6 A) suggested
1
KTC
between S172
N-acetyl group of a-galactosamine is shown
dotted line). (C) Melibiose (as an orange
and oxygen of the
(
stick) from the complex with human GH27
a-galactosidase (3HG3) [27] superimposed
on the modelled AglC structure (grey lines).
Suggested hydrogen bonds are shown as
dotted lines.
3
546
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS

H. Nakai et al.
Transglycosylation by A. nidulans a-galactosidase
analysis). The molecular mass of AglC was estimated by
Cloning of aglC and construction of the
SDS ⁄ PAGE stained with Coomassie Brilliant Blue and by
expression plasmid
TM
1
TM
gel filtration (HiLoad
200 Superdex
¨
16 ⁄ 60 column;
flow rate, 0.5 mLÆmin) ; AKTAexplorer; GE Healthcare)
equilibrated with 10 mm MES pH 6.8, 0.15 m NaCl and
using the Gel Filtration Calibration kit HMW (GE Health-
care) as standards.
The gene aglC (GenBank, gi: 40739585) was cloned by
direct PCR [49] from a P. pastoris X-33 transformant har-
bouring the expression plasmid aglC ⁄ pPICZaC (FGSC
database accession no. 10122; http://www.fgsc.net) [12] pur-
chased from Fungal Genetics Stock Center (School of Bio-
logical Sciences, University of Missouri, MO, USA) with
elimination of both the Saccharomyces cerevisiae a-factor
signal peptide [50] and the AglC signal sequence (Met1-
Ala26). The Expand High Fidelity PCR System (Roche,
Basel, Switzerland) was used as DNA polymerase with
oligonucleotides based on the genomic sequence [44]:
Routine enzyme assay
AglC (0.18–0.27 nm) hydrolysed 2 mm pNPaGal in 40 mm
Na acetate pH 5.0, 0.02% BSA (50 lL) for 10 min at
2 3
37 ꢀC. The reaction was stopped by 1 m Na CO (100 lL)
and released pNP was measured spectrophotometrically
1
mM
5
¢-GGGGAGCTCATTGCGCAGGGTACAACTGGTTCC
from the absorbance at 410 nm using E
= 2.01. One
AATG-3¢ containing a SacI site (underlined) as 5¢ forward
and 5¢-CCCTCTAGACTGCCTTTCTAAGAAGACCACT
TTG-3¢ containing an XbaI site (underlined) as the 3¢
reverse primer. The PCR product was purified (QIAquick
Gel Extraction Kit; Qiagen, Germantown, MD, USA),
digested by SacI and XbaI (New England Biolabs, Ontario,
Canada) and cloned into pCold I [47] (Takara, Kyoto,
Japan). The plasmid was propagated in E. coli DH5a
unit of activity was defined as the amount of enzyme that
liberates 1 lmol pNP from pNPaGal per minute under
these conditions.
Characterization of enzymatic properties
The pH optimum of 0.27 nm AglC for 2 mm pNPaGal
was determined in 40 mm Britton-Robinson buffer [51]
(50 lL; pH 2.3–11.9; 40 mm acetic acid, 40 mm phospho-
ric acid, 40 mm boric acid, pH adjusted by NaOH), as
above. The temperature optimum of activity (see above) in
the range 20–90 ꢀC was determined in 40 mm Na acetate
pH 5.0, 0.02% BSA (100 lL). The dependence of AglC
stability on pH and temperature was deduced from resid-
ual activity analysed by the standard assay for 1.6 nm
AglC in 90 mm Britton-Robinson buffer (pH 2.3–11.9),
0.02% BSA incubated at 4 ꢀC for 24 h and for 4.1 nm
AglC in 20 mm HEPES pH 7.0, 0.02% BSA incubated at
20–90 ꢀC for 15 min. Each experiment was carried out in
triplicate.
(Novagen, Madison, WI, USA), purified (QIAprep Spin
Miniprep Kit; Qiagen), and its sequence verified (MWG
Biotech, Ebersberg, Germany).
Recombinant AglC production
Escherichia coli BL21(DE3) (Novagen) harbouring aglC-
pCold I was grown at 12 ꢀC in Luria-Bertani medium
(
1% tryptone, 0.5% yeast extract, 1% NaCl) containing
1
5
0 lgÆmL) ampicillin (2 · 1 L in 2 L shake flasks).
Expression of aglC was induced by 0.1 mm isopropyl-1-
thio-b-galactopyranoside and continued at 12 ꢀC for 24 h.
Cells were harvested by centrifugation (9000 g, 10 min,
Hydrolytic activity of 0.27–270 nm AglC was tested
towards 5 mm pNPaGal, pNPaGalNAc, pNPaAra,
pNPaAraf, pNPaGlc, pNPaGlcNAc, pNPaMan, pNPaRha,
pNPaXyl, and pNPbGal, and 0.4% galactomannans in
40 mm Na acetate pH 5.0, 0.02% BSA, for 10 min at
37 ꢀC.
Initial rates of hydrolysis of 0.10–2.0 mm pNPaGal,
1.0–12 mm melibiose and 1.0–12 mm raffinose were mea-
sured at seven different substrate concentrations using AglC
(0.18 nm for pNPaGal, 0.37 nm for melibiose, 0.91 nm for
raffinose) in 40 mm Na acetate pH 5.0, 0.02% BSA (1 mL)
at 37 ꢀC. Aliquots (100 lL) removed at 0, 5, 10, 20, 30 min
4
ꢀC), resuspended in 20 mL BugBuster Protein Extrac-
tion Reagents (Novagen) containing 2 lL Benzonate
Nuclease (Novagen) followed by 30 min at room tempera-
ture and centrifugation (19 000 g, 15 min, 4 ꢀC). The
supernatant was filtered (acetate, pore size: 0.22; GE
Infrastructure Water & Process Technologies Life Science
Microseparations, Trevose, PA, USA) and applied to
HisTrap HP (5 mL; GE Healthcare UK, Uppsala,
Sweden) equilibrated with 20 mm HEPES pH 7.5, 0.5 m
¨
NaCl, 10 mm imidazole (AKTAexplorer; GE Healthcare)
and washed with 20 mm HEPES pH 7.5, 0.5 m NaCl,
2
2
2 mm imidazole. Protein was eluted by
a
linear
2 3
were mixed with 1 m Na CO (200 lL) for pNPaGal or
2–400 mm imidazole gradient in the same buffer and
2 m Tris-HCl pH 8.0 (200 lL) for melibiose and raffinose
to stop the reaction. pNP was quantified spectrophotomet-
rically as above. Galactose released from melibiose and
raffinose was quantified using the Lactose ⁄ Galactose
AglC-containing fractions were pooled, dialysed against
0 mm HEPES pH 7.0, and concentrated (Centriprep
2
YM50; Millipore Corporation, Billerica, MA, USA). All
purification steps were performed at 4 ꢀC. The protein
concentration was measured spectrophotometrically at
m
(Rapid) kit (Megazyme). K and kcat were determined from
Lineweaver–Burk plots (1 ⁄ s)1 ⁄ v plots). Each experiment
was carried out in triplicate.
0.1%
2
80 nm using E
= 1.44 (determined using amino acid
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS
3547

Transglycosylation by A. nidulans a-galactosidase
H. Nakai et al.
heat inactivation (15 min, 90 ꢀC) and desalting (Amberlite
MB20; Sigma), the products were purified by HPLC
using a TSKgel Amide-80 column at a constant flow of
1.0 mLÆmin) at 70 ꢀC (see Table S4 for mobile phase com-
position). Fractions containing products were collected
(Foxy Jr. Fraction collector; Teledyne Isco, Lincoln, NE,
USA) and the purity verified by TLC.
TLC and HPLC monitoring of transglycosylation
Transglycosylation products formed by AglC (18 nm for
pNPaGal, 37 nm for melibiose, 91 nm for raffinose) with
1
40 mm substrate in 40 mm Na acetate pH 5.0 at 37 ꢀC for
0–1 h were detected by TLC (TLC Silica gel 60 F254; Merck,
Darmstadt, Germany) developed by acetonitrile ⁄ water
85:15, v ⁄ v) – twice for the transglycosylation products from
(
pNPaGal and three times for the products from melibiose
and raffinose, respectively – and sprayed by a-naphthol ⁄ sul-
furic acid ⁄ methanol (0.03:15:85, w ⁄ v ⁄ v) followed by tarring
at 120 ꢀC. Transglycosylation products were separated by
HPLC (UltiMate 300; Dionex, Sunnyvale, CA, USA)
equipped with a TSKgel Amide-80 column (4.6 · 250 mm;
ESI-MS
ESI-MS was performed using an LTQ XL Ion Trap MS
(Thermo Scientific, San Jose, CA, USA) [52]. Samples were
introduced through a Thermo Accela UHPLC system
equipped with a Hypercarb (100 · 2.1 mm, 3 lm) column
(Thermo) eluted by a gradient of deionized water, acetoni-
trile in 0.2% trifluoroacetic acid (0.4 mLÆmin)1; 70 ꢀC). MS
detection was performed in positive mode using a spray
voltage of 4.5 kV and a capillary temperature of 260 ꢀC
and auto-tuned on glucohexaose (m ⁄ z 1013).
Tosoh Bioscience, Tokyo, Japan) at a constant flow of
1
1
.0 mLÆmin) (see Table S4 for mobile phase composition
and column temperature) and quantified from peak areas
detected by an ultraviolet detector (UltiMate 300 series vari-
able wavelength detector VWD-3400; Dionex) or refractive
index detector (Shodex RI-101; Showa Denko K.K.,
Kanagawa, Japan) calibrated with pNPaGal, melibiose and
raffinose for pNP a-galactobioside, galactosyl-melibiose and
galactosyl-raffinose, respectively. Transglycosylation yields
were calculated based on substrate concentration.
NMR analysis
NMR spectra were recorded on a Bruker DRX 600 spec-
trometer (Bruker BioSpin AG, Fa
¨
llanden, Switzerland) in
5
mm NMR tubes at 300K. Relative amounts were
obtained by integration of one-dimensional proton spectra.
A series of two-dimensional homo- and heteronuclear cor-
related spectra were obtained by Bruker standard experi-
ments COSY, NOESY, TOCSY, heteronuclear single
quantum coherence and heteronuclear multiple bond corre-
lation spectra. The following parameters were used: acquisi-
tion time 0.4 s, NOESY mixing time 0.8 s, 0.12 s TOCSY
spinlock and data points 4096*512 with zero filling in F1
dimension.
Transglycosylation with mono- and disaccharide
acceptors
a-Galacto-oligosaccharides after 3 h reaction in mixtures
(1 mL) containing 18 nm AglC, 40 mm pNPaGal, 400 mm
acceptor (Table 3) in 40 mm Na acetate pH 5.0 at 37 ꢀC
were quantified from peak areas in high-performance
anion-exchange chromatography equipped with pulsed
amperometric detection (HPAEC-PAD, ICS-3000 ion
chromatography system; Dionex; linear gradient 0–75 mm
Na acetate in 100 mm NaOH for 35 min; flow rate
)
1
Homology modelling of AglC
0
.35 mLÆmin ) calibrated with melibiose and raffinose for
di- and trisaccharide products, respectively. Transglycosyla-
tion yields were calculated based on pNPaGal concentra-
tion. Aliquots (50 lL) were removed at 0, 2, 4, 6, 8, 10, 20,
The three-dimensional structure of AglC covering Gly193-
Ala347 (b6–b15 of the N-terminal b-sandwich domain) and
Thr348–Glu699 [the (b ⁄ a) -barrel catalytic domain] was
8
30, 60, 120 and 180 min and 0.5 m NaOH (500 lL) was
added to stop the reaction.
modelled (swiss model; http://swissmodel.expasy.org/) [53]
using the structure of GalA (PDB ID: 1ZY9) [24] as a tem-
plate. An equilibrium mixture of a- and b-galactose from
O. sativa a-galactosidase (1UAS) [28], melibiose from
human a-galactosidase (3HG3) [27] and N-acetyl-a-galac-
tosamine from G. gallus a-N-acetylgalactosaminidase
(1KTC) [29] complexes of GH27 were superimposed on to
the modelled AglC, as no GH36 ligand complex structure
is available.
Sample preparation for oligosaccharide structural
analysis
Transglycosylation products for structure determination
were generated by (a) AglC (18 nm for pNPaGal, 37 nm for
melibiose or 91 nm for raffinose) reacting with 40 mm sub-
strate in 40 mm Na acetate pH 5.0 (final volume 1 mL) at
3
7 ꢀC for 1 h and (b) AglC (18 nm) reacting with 40 mm
Acknowledgements
pNPaGal and 400 mm acceptor (Table 3) in 40 mm Na ace-
tate pH 5.0 (final volume 1 mL) at 37 ꢀC for 2 h (with
melibiose and trehalose) or 3 h (other acceptors). Following
Anne Blicher is thanked for amino acid analysis,
Louise Enggaard Rasmussen for maintaining the
3
548
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS

H. Nakai et al.
Transglycosylation by A. nidulans a-galactosidase
P. pastoris transformant strain and Andrew J. Mort
for making the transformant available. Folmer Fredsl-
und is thanked for help with the structural analysis.
Jørn Dalgaard Mikkelsen and Anne S. Meyer are
thanked for discussions. This study was supported by
the Danish Strategic Research Council’s Committee on
Food and Health (FøSu, to the project ‘Biological
Production of Dietary Fibres and Prebiotics’, no.
10 Fridjonsson O, Watzlawick H, Gehweiler A, Rohrhirsch
T & Mattes R (1999) Cloning of the gene encoding a
novel thermostable a-galactosidase from Thermus
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Table S2. H and C NMR data assignment for
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5
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Fig. S1. SDS ⁄ PAGE of recombinant AglC (4.8 lg).
1
13
Table S1. H and C NMR data assignment for
a-galacto-oligosaccharides produced from pNPaGal,
FEBS Journal 277 (2010) 3538–3551 ª 2010 The Authors Journal compilation ª 2010 FEBS
3551