Effects of truncation at the non-homologous region of a family 3 β-glucosidase from Agrobacterium tumefaciens

Article abstract of DOI:10.1271/bbb.68.1113

The function of the non-homologous region of a family 3 β-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants n

Full text of DOI:10.1271/bbb.68.1113

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Effects of Truncation at the Non-homologous Region
of a Family 3 β-Glucosidase from Agrobacterium
tumefaciens
Li YINGab, Motomitsu KITAOKAa & Kiyoshi HAYASHIa
a Enzyme Laboratory, National Food Research Institute
b College of Biological Sciences, China Agricultural University
Published online: 22 May 2014.
To cite this article: Li YING, Motomitsu KITAOKA & Kiyoshi HAYASHI (2004) Effects of Truncation at the Non-homologous
Region of a Family 3 β-Glucosidase from Agrobacterium tumefaciens , Bioscience, Biotechnology, and Biochemistry, 68:5,
1113-1118, DOI: 10.1271/bbb.68.1113
To link to this article: http://dx.doi.org/10.1271/bbb.68.1113
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Biosci. Biotechnol. Biochem., 68 (5), 1113–1118, 2004
Effects of Truncation at the Non-homologous Region
of a Family 3 ꢀ-Glucosidase from Agrobacterium tumefaciens
y
1
Li YING,1;2 Motomitsu KITAOKA,1; and Kiyoshi HAYASHI
1Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2College of Biological Sciences, China Agricultural University, Beijing, 100094, China
Received December 17, 2003; Accepted February 11, 2004
The function of the non-homologous region of a
family 3 ꢀ-glucosidase from Agrobacterium tumefaciens
(Cbg1) was studied by analyzing the properties of
mutant enzymes that have internal truncated amino
acid sequences in the region. Five truncated mutants
named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and
Cbg1-d119 having deletions of 4, 31, 62, 89, and 119
amino acid residues starting from Phe417, respectively,
were expressed in Escherichia coli and purified. All the
mutants exhibited ꢀ-glucosidase activity, indicating that
the non-homologous region was not essential for the
activity. The truncation caused thermal instability,
decrease in pKa of the proton donor residue (Glu616),
and deficient transglycosylation activity. The thermal
stability and the pKa of Glu616 were partially recovered
with longer truncation, suggesting that the truncation
perturbed the structure and that their presence in the
region was not essential. The main role of the non-
homologous region could be formation of a hydrophobic
atmosphere at the acceptor site to make the enzyme
suitable for hydrolyzing hydrophobic glucosides.
In GH3, barley ꢀ-D-glucan exohydrolase is the only
enzyme whose three-dimensional structure is known.4)
The enzyme consists of an N-terminal ðꢁ=ꢀÞ8 TIM barrel
domain and a C-terminal six-stranded ꢀ sandwich,
connected by a helix-like strand of 16 amino acid
residues. The catalytic center is located in the pocket at
the interface of the two domains. Asp285 in the N-
terminal domain acts as a catalytic nucleophile, while
Glu491 in the C-terminal domain acts as a proton
donor.4,5)
Alignment of the amino acid sequences of Cbg1 with
several family 3 ꢀ-glucosidases has shown that these
enzymes consist of three domains—an N-terminal
homologous domain, a non-homologous domain, and a
C-terminal homologous domain.6) Hence the N-terminal
and C-terminal homologous regions of Cbg1 might
correspond to the N-terminal and C-terminal domains of
barley ꢀ-D-glucan exohydrolase. Catalytic residues of
Cbg1 were presumed to be Asp222 (nucleophile) and
Glu616 (proton donor).7) To investigate the role of the
domains on the catalytic characters of the enzymes,
chimeric enzymes were designed and constructed6–9)
from Cbg1, a family 3 ꢀ-glucosidase from Cellvibrio
gilvus (CG-bgl),10) and a family 3 ꢀ-glucosidase from
Thermotoga maritima (TM-bglB).11) Chimeric enzymes
which had their junction in the C-terminal homologous
region were expressed in Escherichia coli as active
soluble enzymes. Most of the chimeric enzymes showed
characteristics that were intermediate between those of
the respective parental enzymes. On the other hand,
none of the chimeric enzymes which had junctions in the
N-terminal homologous region were expressed as
inactive inclusion bodies. But chimeric enzymes with
junctions in the non-homologous region were not
investigated because of the difficulty in designing
junction sites with completely difference sequences.
In the non-homologous region, the length varies with
the enzyme. In particular, Cbg1 has a longer non-
homologous region than do CG-bgl or TM-bgl.6,7) To
investigate the role of the long non-homologous region
in Cbg1, mutant enzymes with truncated amino acid
sequences in the non-homologous region were designed
Key words: family 3 ꢀ-glucosidase; Agrobacterium
tumefaciens; transglycosylation; truncated
mutant
Agrobacterium tumefaciens causes crown gall disease
in conifers. In this disease, ꢀ-glucosidase (Cbg1) in A.
tumefaciens hydrolyzes coniferin to form coniferyl
alcohol, which leads to a virulence cascade.1,2) Based
on its amino acid sequence, Cbg1 belongs to glycoside
hydrolase family 3 (GH3, http://afmb.cnrs-mrs.fr/
CAZY/).2) It is a typical aryl ꢀ-glucosidase, exhibiting
low Km values (0.01–0.06 mM) on various aryl ꢀ-D-
glucopyranosides and a high Km value on cellobiose
(360 mM).3) Cbg1 possesses wide substrate specificity,
being able to hydrolyze various aryl-glycosides of
monosaccharides such as ꢀ-D-xylopyranoside, ꢀ-D-gal-
actopyranoside, and ꢁ-L-arabinofuranoside, with com-
parable kcat values to that of ꢀ-D-glucopyranoside. It
should be noted that Cbg1 exhibits strong transglycosy-
lation activity toward various primary alcohols.3)
y
To whom correspondence should be addressed. Fax: +81-29-838-7321; E-mail: mkitaoka@nfri.affrc.go.jp

1114
L. YING et al.
and expressed in E. coli. We found that the non-
homologous region plays an important role in trans-
glycosylation.
described in lower case letters. A reverse primer, 50-
GTG GCG AGA AAG GAA GGG AAG AAA G-30,
was designed based on the sequence of the pET28a
vector (Novagen, Madison, Wisconsin, USA) at approx-
imately 200 bp downstream of the stop codon of the
cbg1 gene. The plasmid pET28-AT was used as a
template. KOD Dash polymerase (Toyobo, Osaka,
Japan) was used in the PCR amplification. The PCR
was carried out at 98 ꢀC for 180 s followed by 30 cycles
of 98 ꢀC for 30 s and 72 ꢀC for 90 s. The PCR products
and pET28-AT were digested by EcoRI and HindIII, and
ligated by a Ligation High Kit (Toyobo) at 16 ꢀC for
16 h. The resultant plasmids were transformed into
E. coli BL21 by electroporation (E. coli PulserÔ, Bio-
Rad, Hercules, California, USA). The plasmids in the
positive colonies were purified with a QIAprep Spin
Miniprep Kit (Qiagen GmbH, Hilden, Germany). To
confirm the truncated genes, DNA sequencing was done
on an Applied Biosystems 310 Genetic Analyzer with a
Dye Terminator Cycle Sequencing Kit (Perkin-Elmer,
Boston, Massachusetts, USA).
Materials and Methods
Bacterial strain and plasmid. An expression plasmid,
pET28-AT, with an inserted cbg1 gene (M59852), was
prepared as described previously.12) The schematic
structure of pET28-AT is shown in Fig. 1. The plasmid
codes for Cbg1 with an additional N-terminal sequence
(23 amino acids) of His-tag and a thrombin cleavage
site. E. coli BL21-Gold competent cells (Stratagene, La
Jolla, California, USA) were used as the host for gene
expression.
Construction of truncated mutants of Cbg1. Standard
recombinant techniques and standard agarose gel elec-
trophoresis were used.13) As shown in Fig. 1, expression
vectors for the truncated mutants of Cbg1 were
constructed by swapping the EcoRI–HindIII fragment
of pET28-AT with a 50-end-truncated fragment prepared
using a polymerase chain reaction (PCR). To produce
the truncated fragments, the five forward primers were
designed based on the sequences 12-357 base pairs (bp)
downstream of the EcoRI site with additional EcoRI
sequences at the 50-end as shown below: d4fwd (50-GG
gaa ttc CTT CCG TCC GGC GAC CTT GA-30), d31fwd
(50-GT gaa ttc ATC TTC GGC ATG ACC AAT G-30),
d62fwd (50-GT gaa ttc TTT TTT GGA ACC GCG AAC
AGC-30), d89fwd (50-TC gaa ttc GAG GCG CCG AAG
GCC AG-30), d119fwd (50-CG gaa ttc GTC GAA ACC
GCC CGC AAG T-30). Additional EcoRI sequences are
Preparation of the mutants of Cbg1. The positive
transformants were grown in 2 ml of LB broth contain-
ing kanamycin (30 ꢂg/ml) for 12 hr at 30 ꢀC, then
transferred into 100 ml of the same medium containing
0.1 mM (final concentration) isopropyl-thio-ꢀ-D-galacto-
pyranoside and incubated for 16 h at 25 ꢀC. Cells were
harvested by centrifugation and disrupted in 20 mM
phosphate buffer (pH 8.0) by sonication. The crude
extract was obtained after centrifugation at 20,000 g for
15 min. The crude extracts were partially purified by a
Ni-NTA agarose (Qiagen) column equilibrated with
Fig. 1. Construction of the Plasmids Encoding the Truncated Mutants of Cbg1.
A: Schematic structure of pET28AT. T, N, NH, and C represent the regions encoding His-Tag, the N-terminal homologous region, the non-
homologous region, and the C-terminal homologous region respectively. Sites for restriction endonucleases are indicated with the name of the
enzyme. Right-sided arrows represent the forward primers, and the left-sided arrow indicates the reverse primer. Numbers indicate the positions
of amino acid residues. Asp222 and Glu616 indicate the amino acid residues presumed to be a nucleophile and a proton donor respectively. B:
pET28-AT and the PCR fragments digested with EcoRI and HindIII. These fragments were ligated to construct the expression vectors encoding
the truncated mutants.

Internal Truncation Mutants of a Family 3 ꢀ-Glucosidase
1115
20 mM phosphate buffer (pH 8.0) and eluted with the
same buffer containing 500 mM imidazole. The active
fraction was further purified with a Q-Sepharose anion
exchange column (Amersham Biosciences, Piscataway,
New Jersey, USA) eluted with a linear gradient (0 to
500 mM) of NaCl in 20 mM MOPS buffer (pH 6.5). The
purities of the enzymes were analyzed by sodium
dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE),14) followed by staining with Coomassie
Brilliant Blue.
chromatography (TLC) plate of silica gel 60 (Merck,
7.5 cm L ꢂ 5 cm W). The plate was developed with
acetonitrile/water (8/2 v/v), and the products on the
plate were visualized by dipping the plate in 5% sulfuric
acid in methanol and then heating it at 140 ꢀC for 3 min.
Results
Construction, expression, and purification of
truncated Cbg1
Five expression vectors coding truncated ꢀ-glucosi-
dases were constructed by swapping the EcoRI-HindIII
fragment of pET28-AT into truncated DNA fragments.
The nucleotide sequence of each truncated ꢀ-glucosi-
dase gene was confirmed by DNA sequencing. The
deletions were the only mutations found. The enzymes
with deletions of 4, 31, 62, 89, and 119 amino acid
residues in the non-homologous region were named
Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-
d119 respectively. The mutant enzymes were expressed
and purified. All five purified ꢀ-glucosidases gave single
bands on SDS-PAGE with reasonable mobilities. The
molecular masses of Cbg1-d4, Cbg1-d31, Cbg1-d62,
Cbg1-d89, and Cbg1-d119 were 90.1, 87.3, 83.9, 80.8,
and 77.8 kDa respectively (Fig. 2). The amino acid
sequence of the truncated region starting from Phe417 is
shown in Fig. 3.
Measurement of ꢀ-glucosidase assay. In the standard
assay, the enzymatic reaction was carried out in 25 mM
MOPS buffer (pH 6.5) containing 2 mM p-nitrophenyl-ꢀ-
D-glucopyranoside (pNP-Glc) and 0.02% bovine serum
albumin (BSA) at 30 ꢀC for 10 min. The reaction was
stopped by adding an equal volume of 0.2 M glycine-
NaOH buffer (pH 10.5). The concentration of the p-
nitrophenol liberated was determined by its absorbance
at 405 nm, with the molecular extinction coefficient
taken to be 18,000 (Mꢁ1cmꢁ1). One unit of ꢀ-glucosi-
dase activity was defined as the amount of the enzyme
that releases 1 ꢂmol of p-nitrophenol per min under the
above conditions.
To determine thermal stability, enzymes were incu-
bated in 25 mM MOPS buffer (pH 6.5) containing 0.02%
BSA at various temperatures for 30 min, followed by an
activity assay under standard conditions. To determine
optimum pH, the enzymatic reaction was carried out
under standard conditions except that the following
buffers were used: acetate buffer (pH 3.5–5.0), MES [2-
(N-morpholino) ethanesulfonic acid] buffer (pH 5.1–
6.1), MOPS buffer (pH 6.2–8.2), Tris–HCl buffer (pH
8.0–9.0), CHES [2-(N-cyclohexylamino) ethanesulfonic
acid] buffer (pH 9.0–10.1), and CAPS [3-(cyclohexyl-
amino)-1-propanesulfonic acid] buffer (pH 10.2–11.4).
To determine kinetic parameters, reactions at 6
concentrations of pNP-Glc ranging from 0.2 to 5 Km
were carried out in a spectrophotometer equipped with a
sample changer and a temperature controller (DU640,
Beckman Coulter, USA). The reactions were started by
adding enzymes appropriately diluted with 25 mM
MOPS buffer (pH 6.5) containing 0.02% BSA to
reaction mixtures containing appropriate concentrations
of substrate in the same buffer at 30 ꢀC. Their absorb-
ance at 405 nm was monitored every 30 s for 10 min. A
molecular extinction coefficient of 3,290 (Mꢁ1cmꢁ1) was
used to quantify p-nitrophenol at pH 6.5. Values of Km
and kcat were calculated using the nonlinear regression
analysis program, GraFit Version 3 (Erithacus Software,
Ltd., Surrey, UK).
Kinetic parameters
All the purified enzymes showed detectable activity.
The kinetic parameters of the enzymes on pNP-Glc are
summarized in Table 1. The increase in the Km values
and the decrease in the kcat values corresponded with the
increase in the size of the truncation. Drastic increases in
Km were observed, the Km of Cbg1-d31 being 16-fold
that of Cbg1-d4 and the Km of Cbg1-d119 being 23-fold
that of Cbg1-d89. On the other hand, drastic decreases in
kcat were observed, the kcat of Cbg1-d31 and Cbg1-d89
being approximately 1/5 those of Cbg-d4 and Cbg1-d62
respectively.
Transglycosylation. Appropriately diluted enzymes
were incubated in the reaction mixture in 25 mM MOPS
buffer (pH 6.5) containing 0.02% BSA, 20 mM pNP-Glc,
and 200 mM 1-propanol for 5 h at 30 ꢀC. The reaction
mixture (1 ꢂl), in which approximately 20–40% of the
pNP-Glc was cleaved, was spotted on a thin layer
Fig. 2. SDS-PAGE of the Purified Enzymes.
M, Molecular mass standard; 1 and 7, Cbg1; 2, Cbg1-d4; 3, Cbg1-
d31; 4, Cbg1-d62; 5, Cbg1-d89; 6, Cbg1-d119.

1116
L. YING et al.
Fig. 3. Amino Acid Sequence of the Truncated Region.
Fig. 4. Thermal Stability Curves.
The vertical line indicates the starting position of the truncation
(Phe417). Bold characters indicate the truncated sequence in Cbg1-
d119. Arrows indicate the end of the truncation in the mutants.
Prediction of the secondary structures was performed by using
Genetyx Win Ver. 5 software (Genetyx, Tokyo, Japan). Underlined
and italic sequences are predicted to form an ꢁ-helix and a ꢀ-sheet
structures respectively.
The activity of each untreated enzyme was defined as 1. Solid
circle, Cbg1; solid square, Cbg1-d4; solid triangle, Cbg1-d31; open
circle, Cbg1-d62; open square, Cbg1-d89; open triangle, Cbg1-d119.
For experimental details see the text.
Table 1. Kinetic Parameters of the Mutants on pNP-Glc
Km
kcat
(sꢁ1
kcat=Kꢁm1
(sꢁ1mM
)
Enzyme
Cbg1
Cbg1-d4
(mM)
)
0.013
0.030
0.49
0.34
0.39
9.1
95
44
7300
1500
18
Cbg1-d31
Cbg1-d62
Cbg1-d89
Cbg1-d119
9.0
11
32
2.1
4.4
5.5
0.48
Thermal and pH properties
The thermal stabilities of the mutant enzymes were
compared with that of the parental enzyme (Fig. 4).
Deletion of the four amino acid residues caused a drastic
decrease in thermal stability. The parental enzyme,
Cbg1, was stable up to 55 ꢀC, but Cbg1-d4 was stable
only up to 25 ꢀC for 30 min. But more deletion caused
partial recoveries in thermal stability. Cbg1-d31 and
Cbg1-d62 were stable up to 30 ꢀC, and -d89 was stable
up to 35 ꢀC.
Fig. 5. pH-Activity Curves.
The highest activity of each enzyme was defined as 1. Solid circle,
Cbg1; solid square, Cbg1-d4; solid triangle, Cbg1-d31; open circle,
Cbg1-d62; open square, Cbg1-d89; open triangle, Cbg1-d119. For
experimental details see the text.
The pH properties of Cbg1 and the mutants are shown
in Fig. 5. The optimum pH values were 7.7 (Cbg1), 7.5
(Cbg1-d4), 5.7 (Cbg1-d31), 5.2 (Cbg1-d62), 6.2 (Cbg1-
d89), and 6.6 (Cbg1-d119).
Transglycosylation activity
The reaction products of the pNP-Glc reaction in the
presence of 1-propanol were identified on TLC. As
shown in Fig. 6, the reactions with Cbg1 and Cbg1-d4
formed propyl-glucoside but no detectable glucose. But
Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 pro-
duced glucose and no detectable propyl-glucoside. This
difference was not caused by secondary hydrolysis of
the transglycosylation product, because each reaction
mixture was analyzed before half of the starting
substrate (pNP-Glc) was consumed. These results
Fig. 6. TLC Analysis of the Reaction Products in the Presence of
1-Propanol.
1, Glc (standard); 2, pNP-Glc (standard), 3, Cbg1; 4, Cbg1-d4; 5,
Cbg1-d31; 6, Cbg1-d62; 7, Cbg1-d89; 8, Cbg1-d119.
clearly indicate that deletion of more than 31 amino
acid residues caused a deficiency in tranglycosylation
activity.

Internal Truncation Mutants of a Family 3 ꢀ-Glucosidase
1117
Discussion
sequence analysis predicted that most of the region
(Leu426–Glu446) formed an ꢁ-helix structure as shown
in Fig. 3. Thus, it is suggested that the ꢁ-helix plays an
important role in maintaining a strong affinity toward
hydrophobic compounds at the acceptor site.
The length of the non-homologous region of Family 3
ꢀ-glucosidase varies with the enzyme. But the effect of
length on enzymatic characteristics is still unclear. Cbg1
possesses a longer non-homologous region than other
enzymes. To investigate the role of the long non-
homologous region located at the center of Cbg1, five
truncated mutants were constructed and expressed. The
non-homologous region was considered to be located
between the N-terminal ðꢁ=ꢀÞ8 TIM barrel domain and
the C-terminal six-stranded ꢀ sandwich, with the
catalytic center being at their interface.4,5,7) All the
truncated mutants prepared in this study were catalyti-
cally active, indicating that the non-homologous region
is not essential for the expression of ꢀ-glucosidase.
However, truncation resulted in shifts in optimum pH,
decreases in thermal stability, and decreases in trans-
glycosyl activity.
The truncated mutants showed lower optimum pH
than did Cbg1. The pH-activity curves narrowed with
truncation (Fig. 5). The narrow curve was most pro-
nounced in the case of Cbg-d62. The widths at the mid-
points of the curves were approximately 4.5 (Cbg1), 4.2
(Cbg1-d4), 3.2 (Cbg1-d31), 1.5 (Cbg1-d62), 2.5 (Cbg1-
d89), and 3.3 (Cbg1-d119). These curves are narrow
only on the basic side. There was no significant
expansion on the acidic side. The acidic and basic sides
of the pH-activity curve are regulated by dissociation/
association of the nucleophile residue and the proton
donor residue respectively. This explains the shift in
optimum pH with decreases in the pKa of the proton
donor Glu616.
When the truncation was long, the thermal stability
and pH optimum partially recovered. These results
strongly suggest that the changes in the properties were
due to structural perturbation caused by the truncation,
rather than by a direct role of the non-homologous
region.
Cbg1 exhibits a very low Km value toward pNP-Glc.
The high transglycosyl activity on hydrophobic alcohols
suggests that the enzyme has a strong affinity toward
hydrophobic compounds at the acceptor site. The
truncation caused an increase in the Km values of
pNP-Glc and also a deficiency in transglycosylation.
The transglycosylation was not recovered by the longer
truncation, suggesting that this is the role of the non-
homologous region.
Acknowledgments
We are grateful to the United Nations University and
Kirin Brewery Company for granting a UNU–Kirin
Fellowship to LY.
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