866
W. Saburi et al. / FEBS Letters 589 (2015) 865–869
of amino acid residues should determine their specificity for the
glucosidic linkage. The amino acid residue next to the catalytic
nucleophile is considered to be the primary determinant for the
the catalytic nucleophile. This study describes the conversion of
selectivity of glucosidic linkage in SmDG from -(1 ? 6)-linkage
to -(1 ? 4)-linkage through site-directed mutations at Val195,
Lys275, and Glu371 in an effort to understand the structural ele-
a
a
enzyme’s specificity [4,5].
Ala or Thr at this position, whereas Val is conserved in the
-(1 ? 6)-specific glucosidases (Table 1). Mutant enzymes of a-
a-(1 ? 4)-Specific glucosidases have
ment which contributes to the a-(1 ? 6)-linkage specificity.
a
(1 ? 6)-specific glucosidases, in which the conserved Val and its
neighboring amino acid residues were mutated, hydrolyzed the
2. Materials and methods
a
-(1 ? 4)-glucosidic linkage, but the mutants retained hydrolytic
activity toward the -(1 ? 6)-linkage in all the cases [4,5]. This
suggests that other important amino acid residues (i.e., structural
2.1. Preparation of mutant SmDGs
a
Site-directed mutagenesis was introduced by the megaprimer
PCR method [10], in which the expression plasmid for wild-type
SmDG [6] was used as a template. Recombinant enzyme was pro-
duced in Escherichia coli BL21 (DE3)-CodonPlus™ RIL (Stratagene;
La Jolla, CA) on a 1 L scale, and purified to homogeneity by
Ni-chelating column chromatography as described previously [7].
The concentration of the mutant enzymes prepared was deter-
mined by amino acid analysis.
elements) involved in the recognition of
present.
DG from Streptococcus mutans (SmDG) is a typical
linkage specific exo-glucosidase. Both SmDG and O16G prefer short
a-(1 ? 6)-linkage are
a-(1 ? 6)-
isomaltooligosaccharides, isomaltose [
a
-
-
D
D
-glucopyranosyl-(1 ? 6)-
-glucopyranosyl-(1 ? 6)-
a-D
-glucopyranose] or isomaltotriose [
-glucopyranosyl-(1 ? 6)-
a
a-D
a-D-glucopyranose], but SmDG has
higher activity toward long-chain substrates than O16G [6]. SmDG
catalyzes transglucosylation at high substrate concentrations to gen-
2.2. Enzyme assay
erate an
a-(1 ? 6)-glucosidic linkage. Transglucosylation was
enhanced by the replacement of the catalytic nucleophile aspartyl
residue with cysteine sulfinate [7]. SmDG is composed of three
In a standard enzyme assay, the reaction velocity for the release
of p-nitrophenol from 2 mM p-nitrophenyl
a-D-glucoside (pNPG,
domains commonly found in GH family 13 enzymes [8]. The b ?
Loop 8 of domain A contains three -helices (A
80, A 80, and
8000), and contributes to the formation of the pocket-shaped sub-
strate binding site. One calcium ion, which is tightly coordinated
by the amino acid residues on the b ? loop 1 of domain A
(Asp21, Asn23, Asp25, Ile27, and Asp29), is predicted to enhance
the thermostability of SmDG [9]. The short b ? loop 4 of domain
A and Trp238 located at the C-terminal of b ? loop 5 are impor-
a
Nacalai Tesque, Kyoto, Japan) was measured as described previous-
ly [6]. The optimum pH was determined from the enzyme activity
at various pH levels. To vary the reaction pH, 40 mM Britton
Robinson buffer (pH 3.5–11) was used as the reaction buffer. The
selectivity of glucosidic linkage was investigated based on the rate
of hydrolysis of a series of glucobioses at 1 mM. A reaction mixture
a
a
a
Aa
a
a
a
(50 lL), containing an appropriate concentration of enzyme, 1 mM
substrate, 40 mM sodium acetate buffer, and 0.2 mg/mL bovine
serum albumin, was incubated at 37 °C for 10 min. The pH of the
reaction buffer was 6.0, but was 5.6 for the Val195 variants (pH
6.0 for only V195I and V195L), K275A, V195A/K275A (VK), and
V195A/E371A (VE). Isomaltose (Tokyo Chemical Industry, Tokyo,
tant determinants for the high preference for long-chain substrate
[6]. The structure of an inactive SmDG mutant (general acid/base
mutant, E236Q) in complex with isomaltotriose occupying the
ꢀ1 to +2 subsites revealed that Lys275 and Glu371 form hydrogen
bonding interactions with the 2OH and 3OH groups of a glucosyl
residue in the +1 subsite [8]. Both the amino acid residues are
Japan), maltose
Nacalai Tesque], kojibiose
glucopyranose, Wako Pure Chemical Industries, Osaka, Japan], niger-
ose -glucopyranosyl-(1 ? 3)- -glucopyranose, Wako Pure
Chemical Industries], and trehalose -glucopyranosyl
glucopyranoside, Nacalai Tesque) were used as the substrates. The
[a-
D-glucopyranosyl-(1 ? 4)-a-D-glucopyranose,
[a-
D
-glucopyranosyl-(1 ? 2)-
a-D-
almost completely conserved in the
exo-glucosidases, whereas these amino acid residues are not pre-
sent in -(1 ? 4)-specific enzymes (Table 1). Hence we predict
that Lys275 and Glu371 are important for hydrolytic activity
a-(1 ? 6)-linkage specific
[a-D
a-D
a
(a-D
a-D-
toward
a-(1 ? 6)-linked substrates together with Val195 next to
Table 1
Multiple-sequence alignment of GH family 13 exo-glucosidases.
Enzyme
Origin
Sequence
Dextran glucosidase
Streptococcus mutans (SmDG)
Lactobacillus acidophilus
190 GFRMDVIDMI
194 GFRMDVIELI
233 TVGETWGAT
237 TVGETWNAT
267 LQHKPE–APKWDYVKELNV
271 LDQQPG–KEKWD-LKPLDL
364 LNELDDIESLN-Y
367 IDEVEDIESINMY
Oligo-1,6-glucosidase
Bifidobacterium adolescentis
Bifidobacterium breve (Agl1)
Bifidobacterium breve (Agl2)
Bacillus cereus
Bacillus coagulans
Bacillus subtilis
Bacillus sp. F5
Geobacillus thermoglucosidasius
Saccharomyces cerevisiae (Ima1)
217 GFRMDVITQI
220 GFRMDVITLI
219 GFRMDVITLI
195 GFRMDVINFI
195 GWRMDVIGSI
195 GWRMDVIGSI
194 GWRMDVIGSI
195 GFRMDVINMI
211 GFRMDVGSLY
287 NVGEAPGIT
290 TVGEAPGIT
289 TVGEAPGIT
252 TVGEMPGVT
252 TVGEAIGSD
252 TVGEANGSD
251 TVGEAGGSD
253 TVGETPGVT
274 TVGEMQHAS
321 IDQE——GSKWN-TVPFEV
324 FDCD——GVKWK-PLPLDL
323 VDQTP—ESKWD-DKPWTP
286 LDSGE—GGKWD-VKPCSL
286 VDTKPGSPAGKWA-LKPFDL
286 IDKEQNSPNGKWQ-IKPFDL
285 IDTKQHSPNGKWQ-MKPFDP
287 LDSGP—GGKWD-IRPWSL
308 VGTSP—LFRYN-LVPFEL
416 LEQYRDLEALNGY
419 LDQYRDLESLNAY
419 LDQYRDLESINAY
380 LDEYRDIETLNMY
382 LEEYDDIEIRNAY
382 LEMYDDLEIKNAY
381 LEMYDDLEIKNAY
381 IEDYRDIETLNMY
404 VEKYEDVEIRNNY
a-Glucosidase
Saccharomyces cerevisiae (Mal1S)
Geobacillus stearothermophilus
Geobacillus sp. HTA-462
Halomonas sp. H11
Bacillus sp. SAM1606
Apis mellifera (HBGI)
210 GFRMDTAGLY
195 GFRIDAISHI
195 GFRIDAISHI
198 GFRLDTVNFY
210 GFRMDVINAI
226 GFRIDAVPHL
219 GFRIDAINHM
219 GFRVDALPYI
273 TVGEVAHGS
253 TVGEANGVT
253 TVGEANGVT
268 TVGEIGDDN
268 TVGETGGVT
296 LLTEAYSSL
289 ILTEAYTEF
283 MLIEAYTNL
305 VGTSP—FFRYN-IVPFTL
401 IEKYEDVDVKNNY
377 IRDYRDVAALRLY
377 IRDYRDVSALRLY
377 —EADVPFERIQ
396 IDEYRDVEIHNLW
403 IYKY-DV————
398 YQETVDPAGCNAG
392 WEDTQDPQGCGAG
287 LWKRK—AD———GSIDV
287 LWERR—AD———GSIDV
302 MPHSAS————————————
302 IDATD—GDKWR-PRPWRL
315 SNVPFN-FKFITDANSSSTP
308 STVPFN-FMFIADLNNQSTA
302 ADFPFN-FAFIKNVSRDSNS
Apis mellifera (HBGII)
Apis mellifera (HBGIII)
Amino acid residues of SmDG mutated in this study. The corresponding amino acid residues of the related enzymes are shaded.