Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities.. When the con-sensus region II, which is one of the
Trang 1Val216 decides the substrate specificity of a-glucosidase
Keizo Yamamoto1, Akifumi Nakayama2, Yuka Yamamoto1,* and Shiro Tabata1
1
Department of Chemistry, Nara Medical University, Japan;2Nara Prefectural Institute for Hygiene and Environment, Japan
Differences in the substrate specificity of a-glucosidases
should be due to the differences in the substrate binding and
the catalytic domains of the enzymes To elucidate such
differences of enzymes hydrolyzing a-1,4- and
a-1,6-glu-cosidic linkages, two a-glucosidases, maltase and isomaltase,
from Saccharomyces cerevisiae were cloned and analyzed
The cloned yeast isomaltase and maltase consisted of 589
and 584 amino acid residues, respectively There was 72.1%
sequence identity with 165 amino acid alterations between
the two a-glucosidases These two a-glucosidase genes
were subcloned into the pKP1500 expression vector and
expressed in Escherichia coli The purified a-glucosidases
showed the same substrate specificities as those of their
parent native glucosidases Chimeric enzymes constructed
from isomaltase by exchanging with maltase fragments were
characterized by their substrate specificities When the
con-sensus region II, which is one of the four regions conserved in family 13 (a-amylase family), is replaced with the maltase type, the chimeric enzymes alter to hydrolyze maltose Three amino acid residues in consensus region II were different in the two a-glucosidases Thus, we modified Val216, Gly217, and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis The Val216 mutant was altered
to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity, indicating that Val216 is an important residue discriminating the a-1,4- and 1,6-glucosidic linkages of substrates
Keywords: family 13; a-glucosidase; Saccharomyces cere-visiae; site-directed mutagenesis; substrate specificity
Glucosyl hydrolases (EC 3.2.1.-) are key enzymes of
carbohydrate metabolism that were found in the three
major kingdoms, and are categorized into 57 structural
families [1,2] Family 13 (a-amylase family) includes
enzymes such as a-amylase, a-glucosidase, pullulanase,
cyclodextrin glucanotransferase, and 1,4-a-D-glucan
branching enzyme, specifically acting on a-1,4- and
a-1,6-O-glucosidic linkages [1] Many primary structures
of the members of family 13 from various origins are now
available, and have been compared to each other The
existence of four highly conserved regions (regions I–IV)
and three acidic residues located in the conserved regions as
catalytic residues has been reported [3–7] Furthermore,
computing secondary structure analysis indicated that
specific structural features of the catalytic (b/a)8-barrel
domain exist in these enzymes [8–10]
The relationship of sequence and structure to substrate
specificity in family 13 enzymes, particularly a-amylase,
cyclomaltodextrinase, and neopullulanase, has been well
studied [11–13] Despite the fact that many a-glucosidases with diverse substrate specificities have been purified and cloned from mammals, plants, and microorganisms, it is still not clear which amino acid residues of a-glucosidase recognize the difference between a-1,4- and a-1,6-glucosidic bonds contained in saccharides
Yeast contains two a-glucosidases, a-1,4-glucosidase (E.C 3.2.1.20, maltase) and oligo-1,6-glucosidase (E.C 3.2.1.10, isomaltase), which act preferentially on maltose or isomaltose and methyl a-D-glucopyranoside (a-mg), respectively The expression of these enzymes is controlled by different polymeric genes, MAL or MGL, separately [14–16] Maltase (the MAL6 product of Saccharomyces carlsbergensis) preferentially hydrolyzed maltose but neither isomaltose nor a-mg, whereas isomaltase hydrolyzes isomaltose and a-mg but not maltose [17,18] Thus, we focused on the structure–function relationship of the two a-glucosidases from Saccharomyces as a model in respect of the difference in their substrate specificities The yeast genome directory which was constructed by Goffeau et al revealed the existence of many homologous open reading frames of a-glucosidase [19] The complete nucleotide sequence of the MAL gene of Saccharomyces has been determined [20], whereas it is not clear which open reading frame corresponds to the MGL gene
In this study, we cloned the genes encoding isomaltase and maltase by means of a RT-PCR method and expressed them in Escherichia coli Subsequently, from a comparison
of the primary structures of the two a-glucosidases, chimeric enzymes were constructed by exchange parts of maltase and isomaltase genes including any one of the four conserved
Correspondence to K Yamamoto, Department of Chemistry, Nara
Medical University, Shijo, Kashihara, Nara 634–8521, Japan.
Fax/Tel.: +81 744 29 8810, E-mail: kama@naramed-u.ac.jp
Abbreviations: a-mg, methyl a- D -glucopyranoside; a-pNPG,
p-nitro-phenyl a- D -glucopyranoside.
Enzymes: a-1,4-glucosidase (maltase) (E.C 3.2.1.20);
oligo-1,6-glucosidase (isomaltase) (E.C 3.2.1.10).
*Present address: Department of General Medicine, Nara Medical
University, Japan.
(Received 2 April 2004, revised 17 June 2004, accepted 5 July 2004)
Trang 2regions for family 13 members In addition, we constructed
mutants of isomaltase by replacing three amino acid
residues after Asp215 in consensus region II with residues
of the maltase type using site-directed mutagenesis The
substrate specificities of all mutant enzymes were examined
We found that one amino acid residue in consensus region
II decided the substrate specificity of isomaltase
Materials and methods
Materials
The yeast strains used were Saccharomyces cerevisiae
D-346 (ATCC 56960) and 727–14C (ATCC 56959) The
bacterial strains and plasmids used were Escherichia coli
JM109, KP3998 [21], pUC18 and pKP1500 [21]
Hydroxyapatite (Gigapite) was purchased from
Seika-gaku Kogyo and hydroxyapatite (Micro-Prep Ceramic
Hydroxyapatite, type I) was from Bio-Rad Maltose,
isomaltose, a-mg, and p-nitrophenyl a-D-glucopyranoside
were from Nakalai Tesque, Japan A site-directed
muta-genesis kit (QuickchangeTM) was obtained from Stratagene
and the bicinchoninic acid protein assay reagent was from
Pierce Chemicals La-Taq polymerase was purchased from
Takara Syuzo and restriction endonucleases and T4 DNA
ligase were from Takara Syuzo, or New England Biolabs
Reverse transcriptase was used from the ExpandTM
Reverse Transcriptase kit from Boehringer Mannheim
The Marathon kit was purchased from Clontech
Labor-atory Oligonucleotides were synthesized by Takara Syuzo
Custom Service
Assay method for enzyme activity a-glucosidase activity
was determined by measuring the release of p-nitrophenol
from p-nitrophenyl a-D-glucopyranoside (a-pNPG)
accord-ing to the method described previously [22] When maltose,
isomaltose, and methyl a-D-glucopyranoside (a-mg) were
used as substrates, the enzyme activity was determined as
the rate of hydrolysis of the substrate by measuring the
release of glucose according to the enzymatic method of
NADP+reduction using hexokinase and
glucose-6-phos-phate dehydrogenase [23]
Cloning of the isomaltase gene fromS cerevisiae
The production of maltase and isomaltase of S cerevisiae
was induced by adding maltose and a-mg to the culture
medium, respectively [22] In the case of the cloning of the
isomaltase gene, total RNA was prepared from S cerevisiae
D-346 grown on a medium including 3% (w/v) a-mg by the
method of Chomczynski & Sacchi [24] The mRNA was
purified from the total RNA using Oligotex-dT 30 (Super)
(Takara Syuzo) according to the manufacturer’s
instruc-tions Double-stranded cDNA was constructed from
poly(A) RNA with the oligo-dT primer using the ExpandTM
Reverse Transcriptase kit The N-terminal amino acid
sequence, TISSAHPETEPK, which was determined from
purified yeast isomaltase, matched the ORF YGR287c on
chromosome VII of S cerevisiae [19] Therefore, the
iso-maltase gene was amplified from the cDNA library by PCR
using the N-terminal sequence of ORF YGR287c and
oligo-dT as primers The 1.8 kb RT-PCR product was ligated to
plasmid pUC 18 after digestion with SmaI and introduced
into E coli JM109 The insert was sequenced by the dideoxy method [25] using the Dye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems) To verify the 5¢ end sequence, 5¢ RACE was performed using the Marathon kit with the AP1 primer and a gene-specific primer (5¢-AGATTGCCTTTCTACAGTCTTCATTC-3¢) accord-ing to the manufacturer’s protocol The 5¢-RACE product was sequenced by a direct sequencing method
Subcloning into the pKP1500 expression vector Forward and reverse primers were designed based on the 5¢- and 3¢-terminal nucleotide sequences of the isomaltase gene (MGL) for cloning into plasmid pKP1500 The forward primer 5¢-ATGACTATTTCTTCTGCACAT CCAGAGACAGAAC-3¢ contains the initiation codon, while the reverse primer 5¢-CTTTCTGCAGACTCA TTCGCTGATATATATTC-3¢ linked a PstI restriction site
to the termination codon PCR was carried out on the isomaltase gene cloned above PCR products were digested with PstI Simultaneously, pKP1500 was digested with EcoRI and PstI, and then the EcoRI site was blunted by the use of a Blunting kit (Takara Syuzo) The vector and the insert MGL gene were ligated with T4 ligase followed by transformation into E coli JM109 The cells were plated on Luria–Bertani agar supplemented with 40 lgÆmL)1 5-bromo-4-chloro-3-indolyl-a-D-glucopyranoside (Boehrin-ger Mannheim), 50 lgÆmL)1ampicillin, and 1 mM isopro-pyl thio-b-D-galactoside and then one day later several blue colonies appeared One of the clones expressing isomaltase was selected The plasmid containing the isomaltose gene was designated pYIM
Cloning of the maltase gene fromS cerevisiae For cloning of the maltase gene, S cerevisiae 727–14C was grown in medium containing 3% (w/v) maltose The mRNA and cDNA were prepared using the same procedure
as described above
The gene-specific primers were synthesized based on the information of Hong & Marmur [20] The reverse primer was modified by introducing a HindIII site seven bases down-stream from the stop codon PCR with La-Taq polymerase was carried out on the cDNA prepared from S cerevisiae 727–14C The 1.8 kb PCR fragment was digested with HindIII Plasmid pKP1500 was digested with EcoRI and HindIII, and then the EcoRI site was blunted by the use of a Blunting kit (Takara Syuzo) The fragment was inserted into the pKP1500 vector and the resulting plasmid was intro-duced into E coli KP3998 Several transformants containing the 1.8 kb insert were selected and sequenced The plasmid carrying the maltase gene was designated pYMA
Expression of recombinant enzymes inE coli and purification of the enzymes
The E coli transformant carrying pYIM (or pYMA) was inoculated into PYG medium [21] supplemented with
50 lgÆmL)1of ampicillin and incubated at 37C Isopropyl thio-b-D-galactoside (final 1 mM) was added when cell density at A660 reached 0.5 and the culture was further incubated for 12 h
Trang 3Cells were resuspended in 50 mM Tris/HCl buffer
(pH 7.5) and sonicated The cell-free extract was applied
to a QAE-Toyopearl column equilibrated with 50 mMTris/
HCl (pH 7.5) and the column was washed with the same
buffer containing 20 mMNaCl The enzyme was eluted with
a linear gradient of NaCl (20–150 mM) in the same buffer
Active fractions were pooled and applied to a column of
Gigapite equilibrated with 20 mMsodium phosphate buffer
(pH 7.0) The enzyme was eluted with a linear gradient
of sodium phosphate buffer up to 150 mM The active
fractions were collected and dialyzed against 40 mMsodium
phosphate buffer (pH 6.8), then subjected to
chromato-graphy on hydroxyapatite (Micro-Prep Ceramic
Hydroxy-apatite type I) The purified enzyme was eluted at 250 mM
phosphate buffer (pH 6.8) by a linear gradient of
40–320 mMphosphate
Construction of chimeric enzymes from recombinant
maltase and isomaltase
Chimeric enzymes were constructed by exchanging
nucleo-tide fragments between the maltase and isomaltase genes at
a single restriction site on the plasmid or by inserting a
fragment which was introduced at a unique restriction site
by PCR
Chimeric enzymes MAa/IMb and IMa/MAb were
con-structed by exchanging two MunI/BglII fragments of pYIM
and pYMA which were cleaved at single restriction sites
with both of these restriction enzymes The chimeric
enzyme, Mun/Bpu was constructed by inserting a fragment,
which was amplified by PCR with the forward
pri-mer 5¢-AGAAGCCATTGCTGAGCAATTTTTGTTC-3¢
(underlining indicates the Bpu1102I restriction site) and the
reverse primer 5¢-AAAAAGCTTGCACTAATTTTATTT
GAC-3¢ (underlining indicates the HindIII restriction site and
stop codon, respectively) and pYMA as a template, into IMa/
MAb at Bpu1102I/HindIII Other chimeric enzymes, Mun/
Bst, Mun/Pst, and Pst/Bst were constructed by the same
method described for the Mun/Bpu chimera The chimeric
enzymes are shown in a schematic diagram in Fig 2
Site-directed mutagenesis
Site-directed mutagenesis (Asp215fi Ala,Val216 fi Thr,
Gly217fi Ala, and Ser218 fi Gly of isomaltase) was
carried out by the use of the Quick ChangeTMSite-Directed
Mutagenesis kit and DNA from pYIM as a template and
two additional mutagenic oligonucleotide primers for each
amino acid substitution according to the instruction
man-ual The sites to which the mutation was introduced were
sequenced to confirm that only the expected mutation had
occurred
Results and Discussion
Cloning of yeast a-glucosidases
Two a-glucosidase genes, encoding isomaltase and maltase,
were isolated from an S cerevisiae cDNA library using the
PCR technique In the case of isomaltase, the N-terminal
amino acid sequence, TISSAHPETEPK, matched ORF
YGR287c on chromosome VII of S cerevisiae [19]
More-over, six peptides, including the N-terminal amino acid sequence (TISSAHPETEPK, GSAWTFDEK, NGPRI
PWEGR) obtained from the native isomaltase were in accord with their nucleotide sequences of the ORF Based on this information, a 1.8 kb fragment was amplified from the cDNA library by PCR using 5¢-sequence of the ORF and oligo dT as primers, and was inserted into pUC18 Sequen-cing of the 1.8 kb insert revealed an open reading frame of
1770 bp including a stop codon, TGA The 589 amino acid protein deduced from the ORF was completely identical to the amino acid sequence deduced from ORF YGR287c The sequence data for isomaltase is available from the DNA Data Bank of Japan with accession number AB109221
The entire coding region of the insert was amplified by PCR and subcloned into the pKP1500 expression vector, and the resulting plasmid was introduced into E coli JM109 Expression of the gene was screened by a plate assay using 5-bromo-4-chloro-3-indolyl-a-D -glucopyrano-side Several blue colonies were found to hydrolyze a-pNPG The expression of isomaltase in these clones was confirmed by their ability to hydrolyze isomaltose and a-mg but not maltose The plasmid containing the isomaltase gene was designated pYIM
The maltase gene was also isolated from the DNA library
of S cerevisiae by PCR using gene specific primers The amplified 1.8 kb fragment was inserted into plasmid pKP1500 and the resulting plasmid was transformed into
E coli KP3998 DNA sequence analysis of the fragment gave 100% identity to the MAL6 gene [15] The plasmid containing the maltase gene was designated pYMA Figure 1 shows a comparison of amino acid sequences between maltase and isomaltase There is 72.1% of sequence identity with 165 amino acid alterations
Assessment of recombinant enzymes in comparison with native a-glucosidases
We assessed the two recombinant a-glucosidases in terms of substrate specificity and immunological identity and com-pared them to their native enzymes The two recombinant a-glucosidases showed the same substrate specificities as those of their parent glucosidases, namely, maltase hydro-lyzed maltose but not isomaltose and a-mg, whereas isomaltase hydrolyzed isomaltose and a-mg but not malt-ose Upon double immunodiffusion, rabbit antiserum against native isomaltase produced a single precipitation line without spurs with recombinant isomaltase (data not shown) When the two recombinant enzymes reacted with antisera against native maltase and isomaltase, the recom-binant enzymes showed the same dose–response as the native enzymes by antiserum neutralization (data not shown) These results indicate that the two recombinant a-glucosidases are identical to their parent enzymes Substrate specificities of chimeric enzymes The comparison of the primary structures of the members
of family 13 from various origins has revealed the existence
of four highly conserved regions I, II, III, and IV [3–7] Thus, for the design of chimeric a-glucosidases, the a-glucosidase genes in the two plasmids, pYMA and pYIM,
Trang 4were divided into five portions taking into account the four
consensus regions Figure 2 is a schematic representation of
a number of the chimeric enzymes Chimeric enzymes were
characterized based on substrate specificities for maltase,
isomaltase, a-mg, sucrose, and a-pNPG, and the Km for
a-pNPG MAa/IMb and IMa/MAb were constructed by a
recombination of the N-terminal fragment containing
consensus region I of isomaltase and maltase, respectively
The recombination had no effect on either the substrate
specificities or the Kmfor a-pNPG (Table 1) In the Mun/
Bam chimera, the amino acids from 488 to the C-terminus
of IMa/MAb were substituted by the corresponding amino
acids of maltase (residues 485–584) The substitution of the
C-terminal fragment of IMa/MAb also had no effect on the
substrate specificities We further dissected the C-terminal
region of IMa/MAb by preparing chimeras with
switch-over points at residues 332 and 231 (Mun/Bpu and Mun/
Bst, respectively) The specific activity for isomaltose of
Mun/Bpu and Mun/Bst were about 10 and 80 times lower
than that of isomaltase, respectively The Kmfor a-pNPG of
Mun/Bpu was the same as that of isomaltase, whereas the
Kmfor a-pNPG of Mun/Bst was about 50 times lower than
that of isomaltase Thus, fragments including consensus
regions III and IV may affect the substrate affinity of the
a-glucosidases To investigate the role of the fragment
containing consensus region II, two chimeras, Mun/Pst and
Pst/Bst, were constructed In the Mun/Pst chimera, a 27
amino acid fragment of Mun/Bst including consensus
Fig 1 Comparison of amino acid sequences
between maltase and isomaltase Identical and
similar amino acid residues are designated by
* and Æ, respectively Four highly conserved
regions of family 13 are underlined.
Fig 2 Schematic diagram of the chimeric enzymes Isomaltase se-quence is represented as an open bar and maltase sese-quence is repre-sented as a shaded bar MunI, PstI, BstBI, and Bpu1102I are restriction sites used for the construction of chimeric enzymes I, II, III, and IV indicate the location of four highly conserved regions of family 13.
Trang 5region II was replaced by the corresponding fragment of
pYMA The substrate specificities of Mun/Pst changed
completely to those of maltase type However, the
charac-teristics of Pst/Bst which contained only the 27 amino acid
fragment of pYIM in pYMA were the same as those of
Mun/Bst Therefore, these results indicate that the fragment
including consensus region II contributes to the
determin-ation of the substrate specificity of a-glucosidase
Site-directed mutagenesis
There were six amino acid differences between the two
a-glucosidases in the fragment including consensus region
II Three out of the six alterations were similar, thus, we
targeted the other three amino acid residues in consensus
region II for site-directed mutagenesis The Val216, Gly217,
and Ser218 in consensus region II of isomaltase were
substituted to the corresponding amino acid residues of
maltase, Thr, Ala, and Gly, respectively The mutant
enzymes G217A and S218G did not exhibit different
substrate specificity to that of isomaltase but their Kmfor
a-pNPG tended toward maltase (Table 2) Mutant V216T
could hydrolyze the a-1,4-glucosidic linkage retaining the isomaltase type substrate specificity and its hydrolyzing ratio of maltose/isomaltose was 1 : 1 As shown in Table 2, doubly and triply mutated enzymes including V216T (V216T/G217A, V216T/S218G, and V216T/G217A/
Table 1 Substrate specificities of the chimeric enzymes The enzyme was incubated with 0.5 M substrate in 100 lL of 0.1 M sodium phosphate buffer,
pH 7.0 at 30 C for 5 min The reaction was stopped by addition of 100 lL of 0.5 M Tris/HCl buffer, pH 7.5, then released glucose was assayed For a-pNPG, an increase of absorbance at 410 nm was measured in 5 m M a-pNPG in 0.1 M sodium phosphate buffer, pH 7.0 at 30 C.
Enzyme
Specific activity (lmolÆmin)1Æmg)1enzyme)
K m for a-pNPG (m M )
Table 2 Kinetic parameters of wild-type isomaltase and site-directed
mutants The consensus region II of isomaltase was mutated to the
maltase type by site-directed mutagenesis For example, V216T was
made by exchanging Val216 of isomaltase with Thr of maltase.
Enzyme
Specific activity (lmolÆmin)1Æmg)1
a-pNPG (m M ) Maltose Isomaltose
V216T/G217A/S218G 6.18 0.57 0.49
Fig 3 Sequence alignment of a-glucosidases of known substrate spe-cificity in the consensus region II Asp residue of the catalytic nucleo-phile is labeled with an arrow and the next residue is highlighted in bold Shown are: Sce D-346, S cerevisiae isomaltase (this study); Bth,
B thermoglucosidasius oligo-1,6-glucosidase [27]; Bce, B cereus suc-rase-isomaltase [28]; Bco, B coagulans sucsuc-rase-isomaltase [29]; Bsp1, Bacillus sp DG0303 a-glucosidase [30], Bsp2, Basillus sp F5 sucrase-isomaltase [31]; Bfl, B flavocaldarius oligo-1,6-glucosidase [32]; Spn, Streptococcus pneumoniae a-1,6-glucosidase [33]; Bsu, B subtilis suc-rase-isomaltase-maltase [34]; Bsp3, Bacillus sp a-glucosidase [35], Tcu, Thermomonospora curvata a-glucosidase [36]; Sce727–14C, S cerevis-iae maltase (this study); Sca, S carlsbergensis maltase [20]; Cal,
C albicans maltase [37]; Hpo, Hansenula polymorpha maltase [38].
Trang 6S218G) exhibited a change in the hydrolyzing ratio of
maltose/isomaltose to 5 : 1, 3 : 1, and 10 : 1, respectively
These facts indicate that the three residues in consensus
region II, particularly Val, plays an important role in
distinguishing between the a-glucosidic linkages of a-1,4
and a-1,6
McCarter and Withers [26] indicated that Asp214 on
the consensus region II of maltase is the catalytic
nucleophile Because the Asp214 of maltase is equivalent
to the Asp215 of isomaltase, a mutant with the residue
altered to Ala was tested for its activity on a-pNPG
None of the mutants including D215A had activity on
a-pNPG and a-mg although the proteins were detected
with antiserum against isomaltase by immunoblotting
(data not shown) Thus, the Asp215 of isomaltase is one
of three active acidic residues which are completely
conserved in a-glucosidase group
Amino acid sequence alignment
Figure 3 shows the amino acid sequence alignment of the
consensus region II of a-glucosidases of known substrate
specificity In the case of a-glucosidases hydrolyzing the
a-1,6-glucosidic linkage, the amino acid residue following
the catalytic nucleophile is Val On the other hand, the
corresponding residue of a-glucosidases which acting on
the a-1,4-glucosidic linkage but does not a-1,6-linkage is
Thr X-ray crystallographic analysis of B cereus
oligo-1,6-glucosidase revealed that Val200 following the
cata-lytic nucleophile Asp199 locates on the long loop region
followed by Nb4, and the side chain of Val200 faces
toward the inside of the catalytic cleft [39] Figure 4 shows
the hypothetical structure of the active site of S cerevisiae
isomaltase in complex with isomaltose or maltose using
the crystal structure of B cereus oligo-1,6-glucosidase [39]
as the starting model In the case of wild-type isomaltase,
isomaltose fit to the active site, whereas maltose cannot
bind to the active site because the side chain of Val216
interfere with binding of a 4-linked glucose The CG1 of
Val216 is too close to the O3¢ of maltose On the other
hand, both isomaltose and maltose can bind to the V216T mutant because the steric hindrance between OG1 of Thr216 and O3¢ of maltose is canceled by the rotation of the side chain of Thr216 The results indicate that the amino acid residue just after the catalytic nucleophile in consensus region II must be involved in the recognition of a-glucosidic linkages
In conclusion, this work was successful in identifying the region and residue important in the determination of the substrate specificity of a-glucosidases The identification of V216T and doubly and triply mutated enzymes altered in substrate specificity will serve as a basis for progress toward further understanding the structure-function relationship of family 13 a-glucosidases
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