peng$$0106
Protein Engineering vol.10 no.1 pp.81–87, 1997
Glucoamylase mutants in the conserved active-site segment
Trp170–Tyr175 located at a distance from the site of catalysis
Bjarne B.Stoffer1,2, Claude Dupont1,3,
Torben P.Frandsen1, Jan Lehmbeck4 and
Birte Svensson1,5
1Department
of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10,
DK-2500 Copenhagen Valby and 4Fungal Gene Technology, Enzyme
Business, Novo Nordisk, Novo Allé, DK-2880 Bagsvaerd, Denmark
2Present address: Department of Chemistry, Laboratory IV, University of
Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
3Present address: Department de Microbiologie Appliquée, Université du
Québec, Institut Armand-Frappier, Québec, Canada H7N 4Z3
5To
whom correspondence should be addressed
To mimic the structure of the 1.8-fold more active (kcat)
Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA
was subjected to site-directed mutagenesis in the Trp170–
Tyr175 segment of the third of the six well-conserved α →
α connecting loops of the catalytic (α/α)6-barrel. While the
Trp170 → Phe, Gln172 → Asn and Tyr175 → Phe mutants
showed an up to 1.7-fold increased kcat and Gly174 → Cys
GA an ~2-fold reduced kcat towards maltotriose and longer
substrates, Asn171 → Ser, Thr173 → Gly and A.niger wildtype GA had very similar kcat and Km values for the
hydrolysis of isomaltose and the malto-oligosaccharides
of DP 2-7. Crystal structures of pseudotetrasaccharide
inhibitor complexes of Aspergillus awamori var. X100 GA,
which is 94% identical to A.niger GA, indicate that Tyr175
is located at binding subsite 4, while the preceding target
residues and the high-mannose type unit on Asn171 are at
a larger distance from the site of catalysis. The mutations
had a modest effect on thermostability; the temperature
for 50% inactivation, Tm, was thus unchanged for Tyr175
→ Phe GA and reduced by 0.2–2.9°C for the other mutants.
The deletion of the N-linked high-mannose unit—in Asn171
→ Ser and Thr173 → Gly GAs—appeared to be of minor
importance for enzyme activity and thermostability, and
did not increase the sensitivity to proteolysis.
Keywords: active-site mutation/mutational N-deglycosylation/
subsite map/thermostability/3D model
Introduction
Glucoamylase (GA) (1,4-α-D-glucan glucohydrolase; EC
3.2.1.3) catalyzes the release of β-D-glucose from the nonreducing ends of starch and malto-oligosaccharides (Weill
et al., 1954). Fungal GAs are very important in the manufacture
of glucose syrups (Saha and Zeikus, 1989). The active site of
GA has seven consecutive glucosyl binding sites, catalysis
occurs between subsites 1 and 2; and α-1,4 and α-1,6 glucosidic
bonds are hydrolyzed at the same catalytic site (Hiromi
et al., 1966, 1973, 1983; Hiromi, 1970; Sierks et al., 1989;
Fagerström, 1991; Frandsen et al., 1994, 1995).
Six conserved functionally important regions in GAs, of
which the third, Asp162–Gly183 (Aspergillus niger numbering), is very critical in activity, were identified by chemical
modification (Clarke and Svensson, 1984a,b; Svensson et al.,
© Oxford University Press
1990) and alignment (Itoh et al., 1987; Coutinho and Reilly,
1994; Henrissat et al., 1994) coupled with mutagenesis (Itoh
et al., 1989; Sierks et al., 1989, 1990, 1993; Sierks and
Svensson, 1992, 1993). They constitute α → α segments
creating the funnel-shaped active site of the GA (α/α)6-fold
(Aleshin et al., 1992, 1994b; Harris et al., 1993). Tyr175 is at
subsite 4 in the pseudotetrasaccharide GA structures (Aleshin
et al., 1994a; Stoffer et al., 1995), while residues 170–174—
also mutated in this work—are at a greater distance from the
catalytic site.
Rhizopus oryzae GA (Ashikari et al., 1986) has a 1.8 times
higher kcat value for the hydrolysis of maltose compared with
A.niger GA (Ohnishi et al., 1990). To enhance the activity of
the latter enzyme, six mutants (Trp170 → Phe, Asn171 →
Ser, Gln172 → Asn, Thr173 → Gly, Gly174 → Cys and
Tyr175 → Phe) were made to mimic Rh.oryzae GA (Figure 1).
The succeeding sequence, Asp176–Gly183, is identical in the
two GAs and comprises residues implicated in catalysis,
substrate binding, transition-state stabilization and bond-type
specificity (Sierks et al., 1990; Sierks and Svensson, 1992,
1994; Harris et al., 1993; Fierobe et al., 1996). Single mutations
in Asp176–Glu180 and in Val181–Ser185 greatly reduced and
had a modest effect on activity, respectively (Sierks et al.,
1990, 1993; Bakir et al., 1993; Sierks and Svensson, 1994;
Svensson et al., 1995).
After these mutations were made, the first crystal structure
was determined of the catalytic domain (residues 1–471) of
Aspergillus awamori var. X100 GA (Aleshin et al., 1992,
1994b), being 94% identical to A.niger GA (Svensson et al.,
1983; Aleshin et al., 1992). Complexes with acarbose and
D-gluco-dihydroacarbose indicate hydrogen bonds between
sugar OH groups and Arg54, Asp55, Leu177, Trp178, Glu180
and Arg305 involved in binding at subsites 1 and 2; an array
of outer subsites leads into these inner ones (Aleshin et al.,
1994a; Stoffer et al., 1995). The geometry of the general acid
and base catalysts, Glu179 (Sierks et al., 1990; Aleshin et al.,
1992) and Glu400, is excellent for glucoside bond cleavage
and assistance in the nucleophilic attack of water at C1 (Harris
et al., 1993; Frandsen et al., 1994). Tyr311 and Trp120 stack
with sugar at subsites 2 and 3, respectively; nonbonded contacts
also exist to the fourth ring (Stoffer et al., 1995). Sugar units
on Asn171 and Asn395 (Svensson et al., 1983) appear in the
structure as chitobiose, carrying unbranched mannotriose and
branched mannohexaose, respectively (Aleshin et al., 1992,
1994b).
In this study, site-directed mutagenesis was used to obtain
six single-residue replacements guided by the sequence of
Rh.oryzae GA to improve the activity of A.niger GA and
define roles of the conserved Trp170–Tyr175 (Figure 1). This
segment includes a distant substrate binding area (Stoffer et al.,
1995) and the N-linked sugar unit on Asn171 (Svensson et al.,
1983) that may influence the structural integrity near the
catalytic residues (Aleshin et al., 1992).
81
B.B.Stoffer et al.
Fig. 1. Protein sequence alignment of GAs around the mutated Trp170–
Tyr175 region (↓) in A.niger. An, A.niger (Svensson et al., 1983; Boel
et al., 1984); Ao, A.oryzae (Hata et al., 1991); Nc, Neurospora crassa
(Stone et al., 1993); Hg, gla1 from Humicola grisea var. thermoidea
(GeneBank HUIGLA1A accession no. M89475); Hr, Hormoconis resinae
(Joutsjoki and Torkkeli, 1992); Sf, glu1 from Saccharomycopsis fibuligera
(Itoh et al., 1987); Sd, Saccharomyces diastaticus (Yamashita et al., 1985);
Ro, Rh.oryzae (Ashikari et al., 1986); Ct, thermophilic Clostridium (Ohnishi
et al., 1992). GA from A.awamori (Nunberg et al., 1984), A.awamori var.
X100 (Aleshin et al., 1992), A.awamori var. kawachi (Hayashida et al.,
1989) and A.shirousami (Shibuya et al., 1990) are identical to the A.niger
sequence in the region shown. GA gla1 from S.fibuligera (Hostinová et al.,
1991) is identical to the S.fibuligera glu1 sequence in the region shown. GA
from Saccharomyces cerevisiae (Yamashita et al., 1987) is identical to the
S.diastaticus sequence in the region shown. Numbers in parentheses refer to
the sequence number of immature proteins.
Materials and methods
Enzymes and reagents
Restriction endonucleases and T4 DNA ligase were purchased
from Boehringer Mannheim. Acarbose, a generous gift from
Drs D.Schmidt and E.Truscheit (Bayer AG, Wuppertal), was
coupled to EAH–Sepharose 4B (Pharmacia) as described
previously (Clarke and Svensson, 1984b). The glucose oxidase
kit, isomaltose and maltotriose through to maltoheptaose were
obtained from Sigma Chemical Co.; the glucose dehydrogenase
kit and maltose monohydrate were acquired from Merck.
Bacterial strains, plasmids, media and oligonucleotides
Escherichia coli DH5α was used as host for the plasmid
pBS1/– (Stratagene) carrying a 1557 bp HindIII–BamHI
cDNA fragment of the wild-type A.awamori GA gene (pBSGA)
modified according to (Sierks et al., 1989, 1990) and for
plasmid pJaL37 containing the A.niger GA gene (J.Lehmbeck,
unpublished data). A.niger and A.awamori GA genes are
different but encode identical amino acid sequences (Svensson
et al., 1983; Boel et al., 1984; Nunberg et al., 1984). Plasmid
preparation, restriction endonuclease analyses and the transformation of competent cells were performed as described
previously (Sambrook et al., 1989). Oligonucleotides (Table I)
were prepared on an Applied Biosystems DNA synthesizer
model 380A. In mutant oligonucleotides a silent mutation in
the Gln168 codon was introduced (Figure 2) to create a unique
ScaI site used as a selection marker.
Mutant preparation
Mutants were constructed using the splicing by overlap extension (SOE) approach (Horton et al., 1989). PCR (using the
GenAmpDNA amplification kit; Perkin Elmer Cetus) and SOE
were carried out in a Perkin Elmer Cetus thermal cycler for
25 cycles (each consisted of 1 min at 94°C, 2 min at 60°C
and 3.5 min at 72°C) followed by a 5 min incubation at 72°C.
Template and primer (Table I) concentrations were 0.4 nM
82
and 5 µM, respectively. PCR fragments of 201 and 1011 bp
were obtained from the NotI site to the mutated position and
from the mutated position to the BamHI site, respectively.
Next, a 1162 bp fragment was made by PCR using these
fragments as a template and oligonucleotides covering the
NotI and BamHI sites as cassette primers (Table I). The DNA
fragment was recovered from agarose gel using GeneClean
(Bio101), digested with NotI and BamHI, and ligated into
pGSGA. Clones were checked for the introduction of the ScaI
site and subcloned by replacing the wild-type 1557 HindIII–
BamHI fragment in pJaL37. Mutations were verified by sequencing the entire fragment (Applied Biosystems DNA sequencer).
The transformation and expression of glucoamylase mutant
genes were performed as described previously (Frandsen et al.,
1994) using A.niger strain TSA-1 lacking part of the GA
encoding glaA. Protoplasts were co-transformed by mutant
GA plasmid and pToC90 (Christensen et al., 1988), which
contains the Aspergillus nidulans amdS gene encoding an
acetamidase, allowing the selection of transformants using
acetamide as the sole nitrogen source (Corrick et al., 1987).
Four independent transformants of each mutant were grown
in 10 ml YPD (10 g l–1 bacto yeast extract, 20 g l–1 bacto
peptone, 20 g l–1 maltose) for 4 days at 30°C. The amount of
GA was estimated by incubating 10 µl of medium with 10 µl
maltose (20 g l–1) for 30 min at 45°C and testing for glucose
using TES strips (Lilly). The transformants expressing the
highest levels of GA were propagated in a fermentor (8 l) and
GA was purified from culture filtrates by affinity chromatography on individual acarbose–Sepharose columns (Clarke and
Svensson, 1984b). G1 and G2 GA (Svensson et al., 1982,
1983, 1986b) were separated on HiLoad Q-Sepharose; purity
was tested by SDS–PAGE, amino acid and N-terminal sequence
analyses, and the measurement of activity (Stoffer et al., 1993).
Analytical techniques
Protein concentrations were determined spectrophotometrically
using an ε280 for G1 of 1.373105 M–1 cm–1 (Clarke and
Svensson, 1984a); ε280 5 1.313105 M–1 cm–1 was used for
Trp170 → Phe GA (Stoffer et al., 1993).
Enzyme assays
The activity was determined towards maltose through maltoheptaose and isomaltose in 50 mM sodium acetate, pH 4.5
and 45°C at 12 substrate concentrations from 0.1253Km to
83Km. Released glucose was measured by the glucose oxidase
method in microtiter plates (Fox and Robyt, 1991; Palcic
et al., 1993; Frandsen et al., 1994). kcat and Km values were
determined by fitting initial rates to the Michaelis–Menten
equation using the program ENZFITTER (Leatherbarrow,
1987). Individual subsite affinities were calculated from kcat
and Km for maltose through to maltoheptaose, as described
previously (Hiromi, 1970; Hiromi et al., 1973, 1983). The
equation ∆∆G‡ 5 –RTln[(kcat/Km)mutant/(kcat/Km)wild type]
(Wilkinson et al., 1983) was used to calculate the change in
activation energy caused by mutation.
Stability
The thermostability in 50 mM sodium acetate, pH 4.3, was
assessed as described previously (Svensson et al., 1986a;
Stoffer et al., 1993) by incubating a mutant (0.9 µM) for 5 min
at temperatures between 25 and 80°C, followed by activity
measurements towards maltose. Stability to subtilisin type
Novo was followed by SDS–PAGE and activity measurements
as described by Stoffer et al. (1993).
Glucoamylase mutation at distant subsites
Fig. 2. Gene sequence of GA from A.awamori in the Trp170–Tyr175 region. One silent mutation was introduced at residue 168 in all mutant genes (Table I)
to create a selection ScaI restriction site. Trp170 → Phe, Asn171 → Ser, Gln172 → Asn, Thr173 → Gly, Gly174 → Cys and Tyr175 → Phe were constructed
individually by PCR with the replacements indicated in bold.
Table I. Primers used for the construction of mutant GAs in the Trp170 → Tyr175 regiona
Mutant enzyme
Primers
Trp170 → Phe
59-CTG
59-CCA
59-CTG
59-CCA
59-CTG
59-CCA
59-CTG
59-CCA
59-CTG
59-CCA
59-CTG
59-CCA
59-AGT
59-TAA
Asn171 → Ser
Gln172 → Asn
Thr173 → Gly
Gly174 → Cys
Tyr175 → Phe
Cassette primersb
TCG
GAG
TCG
GAG
TCG
GAG
TCG
GAG
TCG
GAG
TCG
GAG
TCA
TAC
TAC
ATC
TAC
ATC
TAC
ATC
TAC
ATC
TAC
ATC
TAC
ATC
ATG
GAC
GTA
ATA
GTA
ATA
GTA
ATA
GTA
ATA
GTA
ATA
GTA
GAA
TCG
TCA
GCT
TCC
GCT
TCC
GCT
TCC
GCT
TCC
GCT
ACA
GCT
TCC
ACG
CTA
CAG
TGT
CAG
TGT
CAG
TGT
CAG
TCC
CAG
TGT
CAG
TGT
AGA
TAG
TAC
CTG
TAC
CTG
TAC
GTT
TAC
TTG
TAC
CTG
TAC
CTG
CTG
GGC
TTC
GTT
TGG
GCT
TGG
GTT
TGG
GTT
TGG
GTT
TGG
GTT
CCT
GAA
AAC
GAA
AGC
CCA
AAC
CCA
AAC
CCA
AAC
CCA
AAC
CCA
ACA
TTC
CAG
GTA
CAG
GTA
AAC
GTA
CAA
GTA
CAG
GTA
CAG
GTA
CTG
GAG
ACA
CTG
ACA
CTG
ACA
CTG
GGA
CTG
ACA
CTG
ACA
CTG
GTT
CTC
GGA
AGC
GGA
AGC
GGA
AGC
GGA
AGC
TGT
AGC
GGA
AGC
CTT
GGT
TAT
TAC
TAT
TAC
TAT
TAC
TAT
TAC
TAT
TAC
TTC
TAC
GGG
ACC
GAT
GTA
GAT
GTA
GAT
GTA
GAT
GTA
GAT
GTA
GAT
GTA
GGC
CGG
CTC TGG-39
CGA CAG-39
CTC TGG-39
CGA CAG-39
CTC TGG-39
CGA CAG-39
CTC TGG-39
CGA CAG-39
CTC TGG-39
CGA CAG-39
CTC TGG-39
CGA CAG-39
GGC CGC-39
GGA-TCC-39
aMutated
bBoth
nucleotides are shown in bold.
cassette primers cover regions in the gene with unique NotI and BamHI restriction sites for subcloning.
Results
Aspergillus culture supernatants contained 3.7–4.2 g l–1 of
mutant and wild-type GAs, except for Trp170 → Phe and
Gly174 → Cys GAs which both produced at ~0.1 g l–1. The
different GA preparations contained 77–89% G1 (residues 1–
616), which was separated from G2 (residues 1–512; Svensson
et al., 1983, 1986b) and used in this study.
Trp170 → Phe, Asn171 → Ser, Gln172 → Asn and wildtype GAs had the same kcat values for the hydrolysis of
isomaltose, while Thr173 → Gly, Gly174 → Cys and Tyr175
→ Phe GAs had slightly higher values (Table II). Compared
with wild type, the kcat value for maltose decreased 10–30%
with all mutants except Tyr175 → Phe GA (Table II). For
longer malto-oligosaccharides, kcat values gained 30–70% in
the case of Trp170 → Phe, Glu172 → Asn and Tyr175 →
Phe, and decreased by 50–70% for Gly174 → Cys GA; all
Km values, and the kcat values of the remaining mutants,
remained essentially unchanged (Table II). Km values increased
significantly only for the hydrolysis of isomaltose by Tyr175
→ Phe GA. The deglycosylated mutants, Asn171 → Ser and
Thr173 → Gly GAs, had their Km values for maltose hydrolysis
reduced by 25%.
Modest improvements in the transition-state stabilization of
isomaltose hydrolysis of ∆∆G‡ 5 –0.5 to –0.9 kJ mol–1
reflected a 25–35% increase in kcat/Km for Asn171 → Ser,
Thr173 → Gly and Gly174 → Cys GAs. Small improvements
of –0.3 to –0.6 kJ mol–1 were observed in the hydrolysis of
maltose, again in the cases of deglycosylated Asn171 → Ser
and Thr173 → Gly GAs, and Tyr175 → Phe GA (Table III).
Gly174 → Cys GA lost 50–60% and Asn171 → Ser lost 25%
in transition-state stabilization (kcat/Km) for longer substrates;
Trp170 → Phe and Glu172 → Asn gained up to 90%, while
Thr173 → Ser and Tyr175 → Phe, dependent on the substrate,
slightly improved or worsened.
The relative specificity for maltose over isomaltose, (kcat/
Km)G2/(kcat/Km)iG2, decreased by 1.6- to 2.0-fold for five
mutants. Only Tyr175 → Phe GA showed a modest, i.e. 1.4fold, increase (Table IV). Furthermore, the selectivity for
maltoheptaose over maltose, (kcat/Km)G7/(kcat/Km)G2, went up
by 1.1- to 1.5-fold for Trp170 → Phe, Asn171 → Ser, Gln172
→ Asn and Tyr175 → Phe GAs, and decreased by ~1.4-fold
for Thr173 → Gly and Gly174 → Cys GAs (Table IV).
Wild-type and mutant GAs show typical subsite maps with
a small negative affinity for subsite 1, a large affinity at subsite
2 and decreasing values from subsites 2 to 7 (Table V). Only
the less active Gly174 → Cys GA had a small positive value
for subsite 1 and a higher affinity at subsite 2 than wild-type.
Wild-type and Tyr175 → Phe GAs have the same thermostability as described by Tm (the temperature of 50% inactivation), while the Tm value of the other mutants decreased by
0.2–2.9°C (Table IV). Moreover, proteolysis of G1 by subtilisin,
resulting in the catalytic domain (residues 1–471; Stoffer et al.,
1993) of the deglycosylated Asn171 → Ser and Thr173 →
Gly GAs, progressed with the same rate and yield as for wild
type (data not shown).
Discussion
A stereoview of GA from A.awamori var. X100 in complex with
the pseudotetrasaccharide inhibitor D-gluco-dihydroacarbose
(Stoffer et al., 1995) outlines the six mutated positions and
83
B.B.Stoffer et al.
Table II. Kinetic parameters for the hydrolysis of isomaltose and malto-oligosaccharidesa by wild-type mutant GAs
Substrate
Trp170 → Phe
Wild-type
kcat
(s–1)
6
6
6
6
6
6
6
0.04b
0.6
0.7
1.4
3.1
5.0
1.6
Isomaltose
Maltose
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
Maltoheptaose
0.41
10.7
33.5
41.3
48.0
56.2
59.7
Substrate
Asn171 → Ser
kcat
(s–1)
6
6
6
6
6
6
6
Isomaltose
Maltose
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
Maltoheptaose
0.46
8.66
24.4
38.2
36.4
40.3
50.3
0.01
0.3
1.1
2.5
0.4
1.9
0.5
Substrate
Thr173 → Gly
kcat
(s–1)
6
6
6
6
6
6
6
0.54
7.64
32.7
50.5
63.8
46.2
61.2
Substrate
Tyr175 → Phe
0.05
0.03
0.5
3.5
2.6
0.4
7.5
kcat
(s–1)
0.53
10.6
38.7
56.4
60.3
62.0
71.0
kcat/Km
(s–1 mM–1)
kcat
(s–1)
19.8 6 2.8
1.21 6 0.14
0.28 6 0.04
0.12 6 0.01
0.11 6 0.01
0.11 6 0.01
0.12 6 0.01
0.021
8.84
120
344
436
511
498
0.46
8.62
44.7
68.5
72.7
70.2
59.2
6
6
6
6
6
6
6
0.05
0.3
1.2
4.3
0.1
2.8
6
6
6
6
6
6
6
6
0.02
0.1
2.3
1.6
2.5
1.1
1.8
Km
(mM)
kcat/Km
(s–1 mM–1)
22.5 6 2.5
1.06 6 0.02
0.36 6 0.02
0.16 6 0.01
0.14 6 0.01
0.12 6 0.02
0.11 6 0.01
0.020
8.13
124
428
519
585
538
Km
(mM)
kcat/Km
(s–1 mM–1)
Gln172 → Asn
Km
(mM)
kcat/Km
(s–1 mM–1)
kcat
(s–1)
17.2 6 1.7
0.84 6 0.03
0.33 6 0.02
0.15 6 0.01
0.11 6 0.01
0.10 6 0.01
0.096 6 0.010
0.027
10.3
73.9
255
331
403
524
0.42
8.82
44.3
68.7
72.3
72.1
68.8
6
6
6
6
6
6
6
0.01
0.20
1.4
3.2
0.8
4.2
0.5
22.1
1.12
0.40
0.13
0.12
0.12
0.15
6
6
6
6
6
6
6
1.5
0.02
0.02
0.02
0.01
0.01
0.01
0.019
7.87
111
528
603
601
459
Gln174 → Cys
Km
(mM)
Isomaltose
Maltose
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
Maltoheptaose
Isomaltose
Maltose
Maltotriose
Maltotetraose
Maltopentaose
Maltohexaose
Maltoheptaose
Km
(mM)
18.0
0.77
0.39
0.17
0.10
0.12
0.18
6
6
6
6
6
6
6
3.8
0.09
0.01
0.01
0.01
0.01
0.01
kcat/Km
(s–1 mM–1)
kcat
(s–1)
0.030
9.92
83.8
297
638
385
340
0.54
9.43
15.7
19.4
22.5
18.9
24.7
Km
(mM)
kcat/Km
(s–1 mM–1)
28.5 6 2.0
0.97 6 0.12
0.35 6 0.02
0.17 6 0.01
0.12 6 0.01
0.13 6 0.01
0.11 6 0.02
0.019
10.9
111
332
503
477
650
6
6
6
6
6
6
6
0.01
0.20
2.7
0.3
2.5
2.4
0.5
Km
(mM)
kcat/Km
(s–1 mM–1)
21.9 6 1.7
1.20 6 0.15
0.36 6 0.01
0.15 6 0.01
0.12 6 0.01
0.13 6 0.01
0.0976 0.020
0.025
7.86
43.6
129
188
145
255
aDetermined
bStandard
in 50 mM sodium acetate, pH 4.5, at 45°C.
deviation.
includes sugar moieties at Asn171 and Asn395 (Figure 3). A
close up (Figure 4) shows the wild-type side chains to be
mutated in Trp170–Tyr175. Tyr175 CE2 is at a distance of
3.44 Å from C6 of the fourth ring of D-gluco-dihydroacarbose.
The remaining mutated residues are further away from the site
of catalysis, but details are lacking of interactions with substrate
at subsites 5–7 because no ligands larger than pseudotetrasaccharides are bound in GA crystals. A separate study on the
significance of enzyme–substrate interactions at a distant
subsite showed a long-range effect on catalysis of the reverse
reaction, i.e. the condensation reaction, for Ser119 → Tyr GA
(Svensson et al., 1995); Ser119 OG hydrogen bonds to OH3
of the fourth ring (Stoffer et al., 1995).
In general, the present mutants have kcat values comparable
with wild-type GA (Asn171 → Ser; Thr173 → Gly) or slightly
increased, typically in the hydrolysis of maltotetraose and
84
longer substrates (Trp170 → Phe; Gln172 → Asn; Tyr175 →
Phe). Because Rh.oryzae GA has a 1.8-fold higher kcat value
towards maltose than A.niger GA, the rationale used in mutant
design was successful on the longer substrates but not on
maltose. However, Km remained mostly at the A.niger GA
wild-type level; thus the slightly weaker binding to Rh.oryzae
GA (Ohnishi et al., 1990) did not recur in the mutants. The
exception was Gly174 → Cys GA, which has an ~2-fold
reduced kcat value. Cys174 presumably influences the structure
near the site of catalysis. Modeling, using the GA-D-glucodihydroacarbose complex (Stoffer et al., 1995), confirms that
the side chain of Cys174 is at an unfavorable van der Waals
distance from Pro122. This may confer structural stiffness and
make the backbone less flexible. Pro122 is near Trp120, which
stacks with the sugar ring at subsite 3 and is critical in catalysis
and transition-state stabilization (Clarke and Svensson, 1984b;
Glucoamylase mutation at distant subsites
Fig. 3. Stereoview of the fold of glucoamylase from A.awamori var. X100 with the pseudotetrasaccharide inhibitor, D-gluco-dihydroacarbose, bound in the
active site (Stoffer et al., 1995). Residues which are discussed in the text are indicated by a number and (1). The carbohydrate moieties of Asn171 and 395
are shown; α-helices are represented by cylinders (Aleshin et al., 1992).
Fig. 4. Stereoview of the mutated region Trp170–Tyr175 of glucoamylase from A.awamori var. X100 containing the pseudotetrasaccharide inhibitor D-glucodihydroacarbose in the active site (Stoffer et al., 1995). Hydrogen bonds are represented by broken lines.
Table III. Increase in activation energy for substrate hydrolysis by mutant
GAs at pH 4.5 and 45°C
Enzyme
Trp170 → Phe
Asn171 → Ser
Gln172 → Asn
Thr173 → Gly
Gly174 → Cys
Tyr175 → Phe
∆∆G‡a (kJ mol–1)
Maltose
Isomaltose
Maltoheptaose
0.221
–0.404
0.307
–0.305
0.311
–0.554
0.129
–0.665
0.265
–0.943
–0.461
0.265
–0.204
–0.135
0.216
1.01
1.77
–0.705
a∆∆G‡ 5 –RTln[(k /K )
cat m mutant/(kcat/Km)wild type].
bNot determined.
Table IV. Tm valuesa and selectivity constants, G2/iG2 and G7/G2b, for wildtype and mutant GAs from A.niger at pH 4.5 and 45°C
Enzyme
Tm (°C)
G2/iG2
G7/G2
Wild-type
Trp170 → Phe
Asn171 → Ser
Gln172 → Asn
Thr173 → Gly
Gly174 → Cys
Tyr175 → Phe
71.7
68.8
71.1
71.5
69.0
69.4
71.7
421
407
381
414
331
314
574
56.3
66.2
50.9
58.3
34.3
32.4
59.2
aTemperature for 50% inactivation.
bG /iG 5 [(k /K )
2
2
cat m maltose/(kcat/Km)isomaltose]
and G7/G2 5 [(kcat/
Km)maltoheptaose/(kcat/Km)maltose].
85
B.B.Stoffer et al.
Table V. Subsite binding energies for the hydrolysis of malto-oligosaccharides (DP 2–7) by wild-type and mutant GAs at pH 4.5 and 45°C
Enzyme
Wild-type
Trp170 → Phe
Asn171 → Ser
Gln172 → Asn
Thr173 → Gly
Gly174 → Cys
Tyr175 → Phe
aNot
Subsite affinity (kJ mol–1)
1
2
3
4
5
6
7
–2.0
–1.4
–2.9
–1.4
–1.5
2.6
–1.7
–20.8
–19.7
–20.6
–19.6
–20.9
–24.2
–20.7
–6.8
–7.2
–5.3
–7.1
–5.7
–4.7
–6.3
–2.8
–3.3
–3.3
–4.1
–3.3
–2.9
–2.9
–0.7
–0.5
–0.9
–0.3
–2.0
–1.0
–1.3
–0.4
–0.8
–0.5
0.0
1.2
0.6
0.2
0.5
0.8
–0.5
0.8
0.4
–l.3
NDa
determined.
Sierks et al., 1989; Olsen et al., 1993; Aleshin et al., 1994a).
Replacing Gly174 with Cys thus, after refinement, results in
distances from Cys174 SG to Pro122 O and C of 2.53 and
2.86 Å, respectively (data not shown). Gly174 is conserved
(Figure 1), and only Rh.oryzae GA has cysteine, which is,
however, preceded by glycine. This region in the GAs thus
seems to be flexible. Furthermore, Gly174 → Cys GA
undergoes a large stability decrease among the present mutants.
We speculate that wild-type activity and stability may be
maintained in the A.niger GA Thr173 → Gly/Gly174 → Cys
double mutant.
The calculated activation energy (∆∆G‡) for the hydrolysis
of isomaltose increased by –0.461 kJ mol–1 to –0.943 kJ mol–1
for Asn171 → Ser, Thr173 → Gly and Gly174 → Cys GAs.
Apparently the active site became more suited for the hydrolysis
of isomaltose upon these replacements. Only for Tyr175 →
Phe was the selectivity for maltose over isomaltose, (kcat/
Km)G2/(kcat/Km)iG2, higher than for wild-type GA because of
decreased and increased ∆∆G‡ values for the hydrolysis of
maltose and isomaltose, respectively. The activation energy
for maltoheptaose was slightly decreased for Trp170 → Phe,
Asn171 → Ser and Tyr175 → Phe GAs, resulting in increased
(kcat/Km)G7/(kcat/Km)G2 selectivity.
Recently, the thermodynamics of acarbose and 1-deoxynojirimycin binding to the present mutants were characterized using
isothermal titration calorimetry (Berland et al., 1995). Although
the Ka value for acarbose (1012 M–1) varied by only 1.4- to
15-fold as a result of the mutations, slightly more favorable
enthalpy was found—at the expense of the entropy contribution—for Gly174 → Cys and Tyr175 → Phe, and considerably
more for Asn171 → Ser, Gln172 → Asn and Gly174 → Cys
GAs (Berland et al., 1995). In excellent accordance with the
location in the 3D structure, the data suggested small longrange changes in the shape or flexibility of the binding pocket.
An indication of how much these changes could alter was
seen for Tyr175 → Phe GA, which is located near subsite 4
and has 10-fold improved affinity for acarbose. Surprisingly,
Tyr175 → Phe has a 1.6-fold lower affinity than wild type for
1-deoxynojirimycin, a glucose analog bound at subsite 1
(Harris et al., 1993). Similarly, mutations in residues 171–173,
far from the catalytic center, decreased the enthalpy of acarbose
binding by 14–21 kJ mol–1 with compensating unfavorable
changes in entropy, suggesting that this remote region influences the ligand complementarity at the level of subsites 1–4
(Berland et al., 1995). The mutation of Asn171 or Thr173
results in loss of the N-linked carbohydrate of Asn171, which
may alter the shape of the binding pocket. This evidence for
long-range structural effects is a good example of the additional
86
information provided by calorimetry over methods that determine only kinetic parameters.
The removal of carbohydrate from Asn171 had no dramatic
effect on the secretion and activity of GA. A minor decrease
in thermostability for Thr173 → Gly GA, but not for
Asn171→Ser, probably originates in that side-chain replacement and not in elimination of the N-linked carbohydrate.
Thus this sugar unit has inferior importance for stability and
activity. In the 3D structure of GA from A.awamori var. X100
(Aleshin et al., 1992, 1994b), the carbohydrate at Asn171 has
a truncated oligomannose branch and makes just a few contacts
with the protein surface far from the active site. Because
Asn171 → Ser and Thr173 → Gly GAs are not more readily
cleaved by subtilisin than wild-type GA, the carbohydrate at
Asn171 also affords no protection against this protease. In
contrast, deglycosylation at Asn395 (shown in Figure 3),
carrying two branches with several interactions with side
chains from α → α segments 1 and 6 near subsite 1 (Aleshin
et al., 1994b), decreased thermostability and increased protease
sensitivity (Chen et al., 1994b).
The extremely well-conserved sequence Asp176–Ser184 in
the third active-site α → α segment of the (α/α)6-barrel has
been investigated extensively by mutagenesis (Sierks et al.,
1990, 1993; Sierks and Svensson, 1992, 1994; Bakir et al.,
1993; Chen et al., 1994a,b; Fierobe et al., 1996). Single
mutants within Val181–Ser184 modulated the activity (Bakir
et al., 1993; Sierks et al., 1993; Chen et al., 1994a,b; Sierks
and Svensson, 1994), while drastic effects in activity and
stability resulted from the mutation of Glu179, the general
acid catalyst (Sierks et al., 1990); Glu180, which is hydrogen
bonded to substrate in subsite 2 (Sierks and Svensson, 1992);
Asp176, involved in stabilization of the backbone of the
reverse turn containing Glu179 (Aleshin et al., 1992; Bakir
et al., 1993; Harris et al., 1993); and the strictly conserved
Leu177 and Trp178 (Sierks et al., 1993). Remarkably, replacement of the entire Val181–Ser184 enhanced activity towards
the α-1,6-linked isomaltose but reduced activity towards α1,4-linked substrates (Svensson et al., 1995). Homolog replacement of Asn181–Asn182–Gly183 by Thr–Tyr–Ala from Hormoconis resinae GA had a modest effect on the activity (Fierobe
et al., 1996).
In conclusion, mutations guided by the Rh.oryzae sequence
in a conserved segment in a distant area of the substrate
binding cleft successfully improved activity 1.7-fold on certain
substrates in accordance with Rh.oryzae GA, having a 1.8
times higher kcat than A.niger GA. The results encourage
engineering of the entire segments in GA by a binding loop
replacement approach (Fierobe et al., 1996) and provide
Glucoamylase mutation at distant subsites
support for future mutational changes and investigations of
substrate binding areas at a distance from the catalytic site in
polysaccharide metabolizing enzymes.
Acknowledgements
D.Boelskifte, S.Ehlers, A.J.Gajhede, B.Corneliussen and L.Christensen are
thanked for excellent technical assistance, Dr M.R.Sierks for supplying the
pBSGA plasmid and Professor R.B.Honzatko for providing the coordinates
of GA from A.awamori var. X100 prior to publication. This work was
supported by the Danish National Agency of Industry and Trade, grant no.
3007, and by the US Department of Agriculture through the Midwest Plant
Biotechnology Consortium.
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Received April 29, 1996; revised September 12, 1996; accepted September
30, 1996
87