The recombinant AglC, produced in high yield 0.65 gÆL1 culture as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with a-1fi 6 regioselectivity from 40 mm 4-nitr
Trang 1hydrolase family 36 catalyses the formation of
a-galacto-oligosaccharides by transglycosylation
Hiroyuki Nakai1, Martin J Baumann1, Bent O Petersen2, Yvonne Westphal3, Maher Abou Hachem1, Adiphol Dilokpimol1, Jens Ø Duus2, Henk A Schols3and Birte Svensson1
1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark
2 Carlsberg Laboratory, Valby, Denmark
3 Laboratory of Food Chemistry, Wageningen University, The Netherlands
Keywords
acceptor specificity; carbohydrate structural
analysis; transglycosylation;
a-galacto-oligosaccharides; a-galactosidase
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
Tel: +45 4525 2740
E-mail: bis@bio.dtu.dk
(Received 17 May 2010, revised 2 July
2010, accepted 5 July 2010)
doi:10.1111/j.1742-4658.2010.07763.x
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 recombinant AglC, produced in high yield (0.65 gÆL)1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with a-(1fi 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-(1fi 6)-d-Galp, a-d-Galp-(1 fi 6)-a-d-Galp-(1 fi 6)-d-Glcp and a-d-Galp-(1fi 6)-a-d-Galp-(1 fi 6)-d-Glcp-(a1 fi b2)-d-Fruf (stachyose), respectively Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol a-d-galactopyranoside (40 mm), a-(1fi 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-rhaml-arabi-nose, 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 (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-(1fi 6)-d-Manp; a-d-Galp-(1 fi 6)-b-d-Glcp-(1 fi 4)-d-Glcp; a-d-Galp-(1fi 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-(1fi 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.
Trang 2a-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-(1fi
6)-d-Glcp], raffinose [a-d-Galp-(1fi 6)-d-Glcp-(a1 fi
b2)-d-Fruf] and stachyose [a-d-Galp-(1fi
6)-a-d-Galp-(1fi 6)-d-Glcp-(a1 fi b2)-d-Fruf], polymeric
galacto-mannans containing a-(1fi 6) linked galactosyl
resi-dues bound to a b-(1fi 4) mannan backbone and
galactolipids [1] a-Galactosidases occur widely in
bac-teria [2–11], fungi [12–15], plants [16,17] and animals
[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
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
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-(1fi 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-(1fi 6)-b-d-Galp-(1 fi 4)-d-Glcp
and a-d-Galp-(1fi 6)-a-d-Glcp-(1 fi 4)-d-Glcp with
melibiose as the donor and sucrose, lactose and
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
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
Results and Discussion
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
Trang 3formation of storage oligosaccharides by
transglycosy-lation using galactinol [O-a-d-Galp-(1fi
1)-l-myo-ino-sitol] as the donor This relationship motivated the
detailed analysis of the capacity and specificity of AglC
in transglycosylation reactions
Overproduction in E coli and purification of AglC
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
(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-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 bacterial a-N-acetylgalactosaminidases [46,47] were monomers
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-arabinoa-d-manno-pyranose (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
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
a-Galactosidase
a-N-Acetylgalactosaminidase
Raffinose synthase
Stachyose synthase
Trang 4at pH 3.6–9.9 (Fig 2B), maximum activity at 50C
(Fig 2C) and retained > 95% activity after 15 min
incubation up to 45C at pH 5.0 (Fig 2D)
AglC hydrolysed the a-galactosidic linkage in
pNPaGal, melibiose [a-d-Galp-(1fi 6)-d-Glcp] and
raffinose [a-d-Galp-(1fi 6)-d-Glcp-(a1 fi b2)-d-Fruf],
but failed to cleave off a-(1fi 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-lytic efficiency (kcat⁄ Km) towards pNPaGal was two
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,
similar to bacterial a-galactosidases [5,6,10,20], whereas plant alkaline a-galactosidase showed 18-fold higher kcat⁄ Kmfor raffinose than melibiose [16]
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) Thsynthas-ese enzymsynthas-es synthsynthas-esize 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-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
20083 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: 32450781), 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: 3237318), 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: 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).
Trang 5sponding to the calculated value of the Na+adduct of
pNP a-d-galactobioside (C18H25NO13+ Na+) 1
H-and13C-NMR spectroscopy indicated the formation of
a single product, pNP a-d-Galp-(1fi 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-(1fi 3) and a-(1 fi 6) linked pNP
a-d-galacto-biosides [33] AglC furthermore synthesized a
tri-(Fig 3B, E) and a tetrasaccharide tri-(Fig 3C, F) from
40 mm melibiose or raffinose in 59 and 37% yields,
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 (C18H32O16+
Na+) and galactosyl-raffinose (C24H42O21+ Na+) and two-dimensional NMR identified the oligosaccha-rides as a-d-Galp-(1fi 6)-a-d-Galp-(1 fi 6)-d-Glcp and a-d-Galp-(1fi 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
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
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 n M
AglC (•) in 40 m M Britton-Robinson buffer
pH 2.3–11.9 (B) pH stability of 1.6 n M AglC (s) in 90 m M Britton-Robinson buffer pH 2.3–11.9 (C) Temperature activity depen-dence for 4.1 n M AglC ( ) at 20–90 C with
10 min reaction (D) Stability of 9.0 n M AglC (h) in the temperature range 20–90 C for
15 min Each experiment was carried out in 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).
Substrate Km(m M ) kcat(s)1) kcatÆKm)1(s)1Æm M )1)
Trang 6of 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)
Progress of transglycosylation during 3 h using
40 mm pNPaGal and 400 mm of the identified 11
func-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
rigorous recognition governs the transglycosylation outcome Analysis of the product structures (see below) accordingly indicated strict a-(1fi 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-(1fi 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-(1fi 6) linked galactosyl-oligosaccharides These
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 a-Galp-(1 fi Glcp (6), galactose (7), sucrose (8), raffinose (9) and a-Galp-(1 fi a-Galp-(1 fi 6)-Glcp-(a1 fi b2)-Fruf (10) are marked by arrows.
Trang 7products include five novel compounds (Fig 5);
a-d-galactopyranosyl-(1fi 6)-d-mannopyranose;
a-d-galactopyranosyl-(1fi 6)-d-glucopyranosyl-(a1 fi
a1)-d-glucopyranose; a-d-galactopyranosyl-(1fi 6)-a-d-gluco
pyranosyl-(1fi 3)-d-fructofuranose; a-d-galactopyr-anosyl-(1fi 6)-b-d-galactopyranosyl-(1 fi 4)-d-fruc-tofuranose; and a-d-galactopyranosyl-(1fi 6)-b-d-glucopyranosyl-(1fi 4)-d-glucopyranose
Substrate recognition by AglC 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 (b⁄ a)8-barrel (Thr348–Glu699) (Fig 6) This truncated 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 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-Table 3 Test of carbohydrate acceptor candidates for
transgly-cosylation as catalysed by AglC using the donor pNPaGal Products
from 40 m M pNPaGal and 400 m M 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).
Yield (%) Monosaccharide
Disaccharide
Melibiose a-Galp-(1 fi 6)-a-Galp-(1 fi 6)-Glcp 6
Maltose a-Galp-(1 fi 6)-a-Glcp-(1 fi 4)-Glcp 46
Trehalose a-Galp-(1 fi 6)-Glcp-(a1 fi a1)-Glcp 26
Sucrose a-Galp-(1 fi 6)-Glcp-(a1 fi b2)-Fruf 28
Turanose a-Galp-(1 fi 6)-a-Glcp-(1 fi 3)-Fruf 26
Lactose a-Galp-(1 fi 6)-b-Galp-(1 fi 4)-Glcp 38
Lactulose a-Galp-(1 fi 6)-b-Galp-(1 fi 4)-Fruf 38
Cellobiose a-Galp-(1 fi 6)-b-Glcp-(1 fi 4)-Glcp 28
Fig 4 Progress of AglC (18 n M ) catalysed transglycosylation with different acceptors (400 m M ) and pNPaGal (40 m M ) 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 m M 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 m M Na acetate in 100 m M NaOH for 35 min; flow rate, 0.35 mLÆmin)1) calibrated with melibiose and raffinose as standards for di- and trisaccharides, respectively.
Trang 8elled 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
(Fig 6B) The a-N-acetylgalactosaminidase from
G gallus, in contrast, has a cavity formed by Ser172
and Ala175 in the (b⁄ a)8-barrel loop connecting b5
and a5 where the N-acetyl group becomes sandwiched
between Tyr176 and Arg197 and Ser172 hydrogen
bonds to the carbonyl oxygen [29]
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
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)
Conclusion
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-(1fi 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
Experimental procedures
Materials
Allose, altrose, galactose, glucose, gulose, mannose,
l-fucose, l-arabinose, l-rhamnose, cellobiose, lactose, lactu-lose, maltose, melibiose, sucrose, trehalactu-lose, 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
Sequence analysis
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
B
C
E
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.
Trang 9software 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
C
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 (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 between S1721KTCand oxygen of the N-acetyl group of a-galactosamine is shown (dotted line) (C) Melibiose (as an orange 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.
Trang 10Cloning of aglC and construction of the
expression plasmid
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]:
5¢-GGGGAGCTCATTGCGCAGGGTACAACTGGTTCC
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
(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 12C in Luria-Bertani medium
(1% tryptone, 0.5% yeast extract, 1% NaCl) containing
50 lgÆmL)1 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 12C for 24 h
Cells were harvested by centrifugation (9000 g, 10 min,
4C), 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, 4C) 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 (A¨KTAexplorer; GE Healthcare)
and washed with 20 mm HEPES pH 7.5, 0.5 m NaCl,
22 mm imidazole Protein was eluted by a linear
22–400 mm imidazole gradient in the same buffer and
AglC-containing fractions were pooled, dialysed against
20 mm HEPES pH 7.0, and concentrated (Centriprep
YM50; Millipore Corporation, Billerica, MA, USA) All
purification steps were performed at 4C The protein
concentration was measured spectrophotometrically at
280 nm using E0.1%= 1.44 (determined using amino acid
analysis) The molecular mass of AglC was estimated by SDS⁄ PAGE stained with Coomassie Brilliant Blue and by gel filtration (HiLoadTM 200 SuperdexTM 16⁄ 60 column; flow rate, 0.5 mLÆmin)1; A¨KTAexplorer; 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
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
37C The reaction was stopped by 1 m Na2CO3(100 lL) and released pNP was measured spectrophotometrically from the absorbance at 410 nm using E1 mM= 2.01 One 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 bophospho-ric acid, pH adjusted by NaOH), as above The temperature optimum of activity (see above) in the range 20–90C 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 4C for 24 h and for 4.1 nm AglC in 20 mm HEPES pH 7.0, 0.02% BSA incubated at 20–90C for 15 min Each experiment was carried out in triplicate
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
37C
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 37C Aliquots (100 lL) removed at 0, 5, 10, 20, 30 min were mixed with 1 m Na2CO3 (200 lL) for pNPaGal or
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 (Rapid) kit (Megazyme) Kmand kcatwere determined from Lineweaver–Burk plots (1⁄ s)1 ⁄ v plots) Each experiment was carried out in triplicate