3-O-α-d-glucopyranosyl-L-rhamnose phosphorylase from Clostridium phytofermentans

Article abstract of DOI:10.1016/j.carres.2011.12.019

We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze α-linked glucobioses, such as trehalose (α1-α1), kojibiose (α1-2), nigerose (α1-3), and maltose (α1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only l-rhamnose as the acceptor among various sugars examined with β-d-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-α-d-glucopyranosyl-l-rhamnose, indicating strict α1-3 regioselectivity. We propose 3-O-α-d-glucopyranosyl-l-rhamnose: phosphate β-d-glucosyltransferase as the systematic name and 3-O-α-d- glucopyranosyl-l-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase.

Full text of DOI:10.1016/j.carres.2011.12.019

Carbohydrate Research 350 (2012) 94–97
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
3-O-a-D-Glucopyranosyl-L-rhamnose phosphorylase from Clostridium
phytofermentans
b,
Takanori Nihira a,b, Hiroyuki Nakai a,b,c, , Motomitsu Kitaoka
⇑
⇑
a Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan
b National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8642, Japan
c Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to
Received 24 November 2011
Received in revised form 15 December 2011
Accepted 19 December 2011
Available online 29 December 2011
glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 pro-
duced in Escherichia coli did not phosphorolyze
1–2), nigerose ( 1–3), and maltose ( 1–4), which are typical substrates for GH65 enzymes. In reverse
phosphorolysis, Cphy1019 utilized only -rhamnose as the acceptor among various sugars examined with
b- -glucose 1-phosphate as the donor. The reaction product was determined to be 3-O- -glucopyranosyl-
-rhamnose, indicating strict 1-3 regioselectivity. We propose 3-O- -glucopyranosyl- -rhamnose:
phosphate b- -glucosyltransferase as the systematic name and 3-O- -glucopyranosyl- -rhamnose phos-
phorylase as the short name for this novel GH65 phosphorylase.
a-linked glucobioses, such as trehalose (a1–a1), kojibiose
(
a
a
a
L
D
a-D
Keywords:
L
a
a-
D
L
3-O-
a-D-Glucopyranosyl-L-rhamnose
D
a-
D
L
phosphorylase
Glycoside hydrolase family 65
Reversible phosphorolysis
Clostridium phytofermentans
Ó 2011 Elsevier Ltd. All rights reserved.
Phosphorylases are exolytic enzymes catalyzing phosphorolysis
of particular glycosides to produce monosaccharide 1-phosphates
with strict substrate specificity. The reaction is reversible, enabling
the practical synthesis of oligosaccharides from a donor monosac-
charide 1-phosphate and any of the several carbohydrate acceptors,
with strict regioselectivity.1,2 Combining two phosphorylases can
often result in the practical preparation of oligosaccharides from
abundantly available natural sugars.2–7 However, there is little var-
iation among phosphorylases, with only 17 known activities.2,8–10
This limits their utilization for the production of oligosaccharides.
Therefore, it would be beneficial to identify phosphorylases with
previously unreported substrate specificities. Screening for new
phosphorylases from natural organisms is difficult because of the
lack of effective screening methods. Two strategies are often used
to enhance the variability of phosphorylases: protein engineering
to change the specificity of a known phosphorylase11–13 and search-
ing for new phosphorylases based on genomic information. Based
on their amino acid sequences, phosphorylases are classified as
members of glycoside hydrolase families (GHs) 13, 65, 94, 112, or
130 (Henrissat, B. personal communication) or glycosyl transferase
families 4 or 35 on Carbohydrate Active Enzymes database (http://
www.cazy.org/).14 Among these families, GH65, GH94, and GH112
are primarily comprised of phosphorylases.
Based on its genomic sequence (GenBank ID: CP000885), we ob-
served that Clostridium phytofermentans possesses a variety of phos-
phorylase homologs belonging to GH65, GH94, and GH112. We
characterized the three GH112 proteins from C. phytofermentans
and found that two of them were 3-O-b-
yl- -hexosamine phosphorylases (EC 2.4.1.211, Cphy0577 and
Cphy3030) and that the third was a 4-O-b- -galactopyranosyl-L-
D-galactopyranosyl-N-acet-
D
D
rhamnose phosphorylase (EC 2.4.1.247, Cphy1920), which had not
been previously reported.8 We also reported that one of the four
GH65 homologs (Cphy1874) was a novel phosphorylase, 3-O-
a-D-glu-
copyranosyl-
D
-glucose phosphorylase (nigerose phosphorylase).10 In
this study, we characterized Cphy1019, another C. phytofermentans
GH65 homolog, with unique substrate specificity.
The amino acid sequence deduced from cphy1019 (756 amino
acids; 87,990 Da) shows no predicted N-terminal signal peptide
based on a SignalP 3.0 analysis (http://www.cbs.dtu.dk/services/
SignalP/),15,16 suggesting that this protein is located in the cyto-
plasm. Although the amino acid sequence of Cphy1019 exhibits
24–29% identity with
a-glucosyl disaccharide phosphorylases
belonging to GH65, such as trehalose,17 kojibiose,18 nigerose,10
maltose,19 and trehalose 6-phosphate phosphorylases,20 this pro-
tein could not be categorized into any of the known GH65 enzymes
based on phylogenetic tree analysis (Fig. 1). Therefore, recombinant
Cphy1019 with additional His6 tag sequence at the C terminal was
produced in Escherichia coli BL21 (DE3) to investigate its enzymatic
properties. Approximately 3 mg of purified protein was obtained
from a 200-mL culture. The purified Cphy1019 showed a single band
in SDS–PAGE with an estimated size of 88 kDa. Cphy1019 did not
⇑
Corresponding authors. Tel.: +81 29 838 8071; fax: +81 29 838 7321.
E-mail addresses: nakai@niigata-u.ac.jp (H. Nakai), mkitaoka@affrc.go.jp
(M. Kitaoka).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.12.019

T. Nihira et al. / Carbohydrate Research 350 (2012) 94–97
95
<Trehalase>
A.nidulans
M.acridum
EAA66407
ABO93464
S.cerevisiae
CAA89280
C. phytofermentans
ABX41399 (Cphy1019)
<Maltose P>
L.acidophilus
AAV43670
C. phytofermentans
ABX42243 (Cphy1874)
<Nigerose P>
Bacillus sp.
BAC54904
Nostoc sp.
BAB76688 *
Paebacillus sp.
BAD97810
Nostoc sp.
BAB73015 *
G.stearothermophilus
T.brockii
BAC20640
AAE30762
T.brockii
AAE18727
C.saccharolyticus
ABP66077
<Trehalose P>
<Kojibiose P>
K.stuttgartiensis
CAJ75349
L.lactis
AAK04526
0.1
<Trehalose-6-P P>
Figure 1. Phylogenetic tree of characterized GH65 enzymes. Multiple alignments were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). A
phylogenetic tree was constructed using TreeView version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html). The gene cloned in this study is represented by the
organism name and GenBank™ accession number with a gray background. The other genes encoding characterized GH65 enzymes (http://www.cazy.org), represented by the
organism names and GenBank™ accession numbers, are categorized in boxes framed with broken lines according to their substrate specificities. ⁄ Characterization of these
proteins were presented by Watanabe et al. and Yamamoto et al. at the annual meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry on March 28,
2010. The abstracts are available on page 23 of the meeting abstracts under paper numbers 2AFa14 and 2AFa15 (in Japanese).
Table 1
NMR analysis of the product
Sugar ring
Position
a
-Anomer (62%)
b-Anomer (38%)
1H NMR
13C NMR
1H NMR
13C NMR
d (ppm)
d (ppm)
Pattern
J (Hz)
d (ppm)
d (ppm)
Pattern
J (Hz)
I (
L
-rhamnose)
1
2
3
4
5
6
1
2
3
4
5
6
95.31
69.52
77.08*
72.11
70.11
18.58
97.28
73.08
74.60
71.00
73.36
61.89
5.14
4.11
3.85*
3.53
3.89
1.28
5.07
3.57
3.78
3.46
3.96
3.78
d, 1H
dd, 1H
dd, 1H
t, 1H
dq, 1H
d, 3H
d, 1H
dd, 1H
m
J1,2 = 1.2
J2,3 = 3.1
J3,4 = 9.7
J4,5 = 9.7
J5,6 = 5.7
95.14
69.16
79.22*
71.79
73.72
18.55
96.99
73.08
74.60
71.00
73.36
61.89
4.85
4.13
3.68*
3.46
3.43
1.30
5.09
3.57
3.78
3.46
3.96
3.78
d, 1H
dd, 1H
dd, 1H
dd, 1H
dq, 1H
d, 3H
d, 1H
dd, 1H
m
J1,2 = 0.7
J2,3 = 3.1
J3,4 = 9.2
J4,5 = 10.1
J5,6 = 5.8
II (
D
-glucose)
J1,2 = 3.8
J2,3 = 9.9
J3,4 = 9.2
J4,5 = 9.9
J1,2 = 3.8
J2,3 = 9.9
J3,4 = 9.2
J4,5 = 9.9
dd, 1H
m, 1H
m, 2H
dd, 1H
m, 1H
m, 2H
NMR spectra are shown in Figures S1–S5. The structure of the compound is shown in Figure 2.
*
Interring HMBC cross peaks were observed with the anomeric position of glucose.
phosphorolyze the
a
-linked glucobioses, such as trehalose (
a
1–
a
1),
acceptor specificity for
ty. We propose 3-O-
b- -glucosyltransferase
L
-rhamnose with strict
-glucopyranosyl-
(3-O- -glucopyranosyl-L
a
-1,3-regioselectivi-
kojibiose ( 1–2), nigerose (
a
a
1–3), and maltose ( 1–4), which are
a
a-
D
L
-rhamnose: phosphate
typical substrates for GH65 enzymes. In reverse phosphorolysis
D
a-
D
-rhamnose
using various carbohydrate acceptors (see Section 1.3) in the pres-
phosphorylase) as the systematic name for Cphy1019 and suggest
that a new EC number is required for this phosphorylase (Fig. 2).
Cphy1019 was stable up to 30 °C for 10 min, and the optimum
temperature for the reverse phosphorolysis was 30 °C (Fig. S6).
This corresponds to the optimum growth temperature of meso-
philic C. phytofermentans.21 Cphy1019 was stable in the range of
pH 6.0–9.0, and the optimum pH for the reverse phosphorolysis
was pH 6.5 (Fig. S6). These temperature and pH profiles suggest
ence of b-
activity only with
product was isolated and identified as 3-O-
D
-glucose 1-phosphate as the donor, Cphy1019 showed
-rhamnose as the acceptor. The disaccharide
-glucopyranosyl-
L
a-
D
L-
rhamnose based on 1H and 13C NMR spectroscopic analyses
(Table 1, Figs. S1–S5). Cphy1019 did not utilize any of the disaccha-
rides examined as acceptors in the reverse phosphorolysis. These re-
sults indicate that Cphy1019 is a novel phosphorylase showing strict

96
T. Nihira et al. / Carbohydrate Research 350 (2012) 94–97
Figure 2. Schematic representation of the reactions catalyzed by 3-O-a-D-glucosyl-L-rhamnose phosphorylase.
that Cphy1019 can function in the cytosol of C. phytofermentans, as
predicted from the amino acid sequence analysis.
(IPTG) when Abs600 reached 0.6 and continued for 6 h. Cells were
harvested by centrifugation at 4000g for 5 min and suspended in
50 mM 3-(N-morpholino)propanesulfonic acid (MOPS)–NaOH (pH
7.5) containing 500 mM NaCl (buffer A). The suspended cells were
disrupted by sonication, and the supernatant collected by centrifu-
gation at 10,000g for 20 min was applied to an immobilized metal
affinity chromatography column equilibrated with buffer A con-
taining 5 mM imidazole using a Profinia protein purification sys-
tem (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After washing
the column with buffer A containing 10 mM imidazole, Cphy1019
was eluted with 50 mM MOPS–NaOH buffer (pH 7.5) containing
500 mM NaCl and 500 mM imidazole, and desalted with 10 mM
MOPS–NaOH (pH 7.5) containing 100 mM NaCl using a Bio-Gel
P-6 column (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The
protein concentration was determined spectrophotometrically at
Kinetic parameters for Cphy1019 with
tor and b- -glucose 1-phosphate as the donor were determined with
10 mM of the other substrates by fitting the data to the Michaelis–
Menten equation (Fig. S7). The apparent Km and kcat values for
rhamnose were 2.7 ( 0.2) mM and 1.6 ( 0.1) sꢀ1, respectively, and
those for b- -glucose 1-phosphate were 9.6 ( 1.9) mM and 2.5
( 0.3) sꢀ1, respectively. The Km value for
-rhamnose is in the same
L-rhamnose as the accep-
D
L-
D
L
range as that of other disaccharide-specific phosphorylases for their
specific acceptors. Although the kcat values of Cphy1019 are much
smaller than that of nigerose phosphorylase from the same organ-
ism,10 they are in the range of kcat values reported for some
phosphorylases.22,23
Because phosphorylases have very strict substrate specificities,
the substrates are presumed to play important roles in organisms
possessing these phosphorylases. Although C. phytofermentans was
280 nm using an extinction coefficient of
1.3. Phosphorolysis and reverse phosphorolysis
Phosphorolysis of -linked glucobioses (trehalose, kojibiose,
e .
= 131,560 cmꢀ1 Mꢀ1
isolated from forest soil, no possible source of 3-O-
a-D-glucopyran-
osyl- -rhamnose was found among plant saccharides. A search of the
L
bacterial carbohydrate structural database (http://www.glyco.a-
c.ru/bcsdb3/)24 found 154 compounds among 9071 registered struc-
tures. Most of these were in repeating unitsof lipopolysaccharides or
capsule polysaccharides. Although no structures of polysaccharides
produced by C. phytofermentans have been identified, the enzyme
may be involved in salvaging such polysaccharides. In conclusion,
a
nigerose, maltose, and isomaltose) was examined at 30 °C in
25 mM MOPS–NaOH (pH 6.5), 10 mM phosphate, 10 mM sub-
strates, and 1.5 lM Cphy1019 by detection of the released glucose
as described previously.10
The reverse phosphorolysis was performed with Cphy1019
we have identified a novel C. phytofermentans 3-O-
a
-
D
-glucopyran-
(1.5
cose 1-phosphate as the donor and 10 mM putative carbohydrate
acceptors [ -rhamnose, -glucose, -mannose, -allose, -galactose,
-xylose, -arabinose, -lyxose, -fucose, -fructose, 2-deoxy- -arabi-
no-hexopyranose (2-deoxy- -glucose), -glucal, -glucosamine,
-glucose 1-phosphate, b- -glucose 1-phosphate, -glucose
6-phosphate, -glucuronic acid, methyl -glucoside, methyl b-
glucoside, 1,5-anhydro- -glucitol, 3-O-methyl- -glucose, 2-acetam-
ido-2-deoxy- -glucose, 2-acetamido-2-deoxy- -galactose, trehalose,
lM) in 25 mM MOPS–NaOH (pH 6.5) containing 10 mM b-D-glu-
osyl- -rhamnose phosphorylase, which is a suitable catalyst for
L
the efficient synthesis of 3-O-
a-D
-glucosyl-
L
-rhamnose with strict
L
D
D
D
D
regioselectivity.
D
L
D
L
D
D
D
D
D
a
-D
D
D
1. Experimental
D
a-D
D-
D
D
1.1. Construction of expression plasmid
D
D
kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose,
cellobiose, gentiobiose, xylobiose, lactose, and sucrose] at 30 °C for
2 h. The reaction mixture (1 lL) was spotted on a thin layer chroma-
tography (TLC) plate (Kieselgel 60 F254; Merck, Darmstadt, Germany),
and the sample was developed with a solution of acetonitrile/water
(4:1, v/v). TheTLC plates werebrieflysoaked in5% sulfuricacid-meth-
anol solution and heated in an oven until bands were sufficiently
visible. The reverse phosphorolytic activity was determined by
measuring the increase in phosphate in the reaction mixture contain-
A gene (cphy1019; GenBank ID: ABX41399.1) encoding a GH65
homolog (Cphy1019) was amplified by PCR with genomic DNA of
C. phytofermentans ATCC700394 as the template. KOD-plus DNA
polymerase (Toyobo, Osaka, Japan) was used with the following
oligonucleotides based on the genomic sequence (GenBank ID:
CP000885): 50-gatatacatatgttaattcatgaagataat-30 as the forward
primer containing an NdeI site (underlined) and 50-ggtgctc-
gaggaaggatacctccaatctgt-30 as the reverse primer containing a XhoI
site (underlined). The amplified cphy1019 was purified with a
MinElute Cleanup Kit (Qiagen, Hilden, Germany) and inserted into
pET24a (Novagen, Madison, WI, USA). The expression plasmid
cphy1019/pET24a was propagated in E. coli XL1 Blue (Agilent Tech-
nologies, Waldbronn, Germany), purified by illustra plasmidPrep
Mini Spin Kit (GE Healthcare UK Ltd, Little Chalfont, Buckingham-
shire, UK), and verified by sequencing (ABI 3730xl sequencer,
Applied Biosystems, Foster City, CA, USA).
ing 10 mM b-D-glucose 1-phosphate and 10 mM L-rhamnose in
25 mM MOPS–NaOH (pH 6.5) at 30 °C by following the method of
Lowry and Lopez25 as described previously.10
1.4. Temperature and pH profile
The effects of pH on the reverse phosphorolytic activity of 1.5 lM
Cphy1019 was measured at 30 °C in 100 mM of the following
buffers: sodium citrate (pH 3.0–5.5), 2-(N-morpholino)ethanesulfo-
nic acid–NaOH (pH 5.5–7.0), 2-[4-(2-hydroxyethyl)piperazin-
1-yl]ethanesulfonic acid–NaOH (pH 7.0–8.5), and glycine–NaOH
(pH 8.5–10.5). The temperature dependence of 764 nM Cphy1019
was measured in 25 mM MOPS–NaOH (pH 6.5) at various tempera-
tures for 10 min. The thermal and pH stabilities were defined as the
1.2. Recombinant Cphy1019 preparation
An E. coli BL21 (DE3) (Novagen, Madison, WI, USA) transformant
harboring cphy1019/pET24a was grown at 25 °C in 200 mL Luria–
Bertani medium containing 33
lg/mL of kanamycin. Expression
was induced by 0.1 mM isopropyl b-
D
-thiogalactopyranoside

T. Nihira et al. / Carbohydrate Research 350 (2012) 94–97
97
reverse phosphorolytic activity remaining after incubation of
Cphy1019 (2.5 M) at various temperatures for 10 min in 25 mM
MOPS–NaOH (pH 6.5) and at various pH values at 30 °C for 30 min,
Universities, France) for helpful discussion on classification of
phosphorylases. This work was supported in part by a grant from
the Program for Promotion of Basic Research Activities for Innova-
tive Biosciences (PROBRAIN) and MEXT’s program ‘Promotion of
Environmental Improvement for Independence of Young Research-
ers’ under the Special Coordination Funds for Promoting Science
and Technology.
l
respectively.
1.5. Kinetic analysis
The reverse phosphorolytic reactions were carried out at 30 °C
with 548 nM Cphy1019 and various concentrations of
L-rhamnose
Supplementary data
(1–40 mM) as the acceptor or b- -glucose 1-phosphate (0.5–
D
10 mM) as the donor with 10 mM of each opposite substrate in
25 mM MOPS–NaOH (pH 6.5). The kinetic parameters were
calculated by curve-fitting the experimental data with the
Michaelis–Menten equation {v = kcat [E]0ꢁ[S]/(Km + [S])} using Grafit
version 7.0.2 (Erithacus Software Ltd, London, UK).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.12.019.
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We thank the staffs of Instrumental Analysis Center for Food
Chemistry of National Food Research Institute for recording NMR
spectra and Dr. Bernard Henrissat (CNRS and Aix-Marseille