Discrimination of aliphatic substrates by a single Amino acid substitution in bacillus and bacillus shaericus phenylalanine dehydrogenases

Article abstract of DOI:10.1271/bbb.80626

Replacement of glycine by serine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius and Bacillus sphaericus respectively strikingly decreased enzyme activity toward aromatic amino acids and resulted in an elevation of relative activity toward aliphatic amino acids. The mutant from B. badius preferentially dehydrogenated branched-chain amino acids, while that from B. sphaericus acted on amino acids with straight-chain amino acids.

Full text of DOI:10.1271/bbb.80626

Biosci. Biotechnol. Biochem., 73 (3), 729–732, 2009
Note
Discrimination of Aliphatic Substrates by a Single Amino Acid Substitution
in Bacillus badius and Bacillus sphaericus Phenylalanine Dehydrogenases
y
Shinjiro TACHIBANA, Yuko KUWAMORI, and Yasuhisa ASANO
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
Received September 4, 2008; Accepted November 19, 2008; Online Publication, March 7, 2009
[doi:10.1271/bbb.80626]
Replacement of glycine by serine at positions 123 and
24 of phenylalanine dehydrogenases from Bacillus
diagnosis of phenylketonuria.10) PheDH from Bacillus
badius has been successfully used in the diagnosis of
1
1
1–13)
badius and Bacillus sphaericus respectively strikingly
decreased enzyme activity toward aromatic amino acids
and resulted in an elevation of relative activity toward
aliphatic amino acids. The mutant from B. badius
preferentially dehydrogenated branched-chain amino
acids, while that from B. sphaericus acted on amino
acids with straight-chain amino acids.
phenylketonuria in Japan for more than a decade.
We have screened for methionine dehydrogenase in
1
4)
nature with limited success, but generation of a novel
amino acid dehydrogenase with substrate specificity for
L-Met might become available for a newborn screening
program for HCU.
Gly-124 of B. sphaericus PheDH is a critical residue
for substrate discrimination. The sequence of B. sphaer-
icus PheDH shows high homology to that of B. badius
PheDH among PheDHs. Especially, position 124 of
B. sphaericus PheDH is entirely identical with position
Key words: phenylalanine dehydrogenase; site-directed
mutagenesis; Bacillus badius; Bacillus
sphaericus
123 of B. badius PheDH. In this study, we constructed
Phenylalanine dehydrogenase (PheDH) [EC 1.4.1.20]
þ
two single mutants, Gly123Ser of B. badius PheDH and
Gly124Ser of B. sphaericus PheDH respectively, by
site-directed mutagenesis, and evaluated the effects of
these replacements on substrate specificity in detail. We
herein report that the replacement of Gly by Ser
dramatically improve enzyme activity toward aliphatic
substrates as compared to single mutant Bs-G124A.
To produce protein fused with 6ꢀ His-tag at the
N-terminus, the pUC18 vector was modified as follows:
The fragment of pRSET-B vector (Invitrogen Co.,
is an NAD -dependent oxidoreductase that catalyzes
the oxidative deamination of L-Phe and the reductive
amination of related ꢀ-keto acid analogs. It belongs to a
family of amino acid dehydrogenases that consists of
glutamate dehydrogenase (GluDH), leucine dehydrogen-
ase (LeuDH), and valine dehydrogenase (ValDH). We
have found that PheDH from Bacillus sphaericus is an
1
)
important catalyst for the synthesis of natural and non-
natural amino acids, some of which are constituents of
2
,3)
0
pharmaceuticals, by the reductive amination reaction.
Based on a molecular modeling using Clostridium
Carlsbad, CA) was amplified by PCR using primers 5 -
0
0
gccgaattcttaagaaggagatatacata-3 and 5 -gctcggatccttatc-
0
4
)
symbiosum GluDH structure (PDB id, 1BGV), Gly-124
and Leu-307 residues in B. sphaericus PheDH have
been proposed to cause the difference in substrate
recognition between PheDH and LeuDH. The corre-
sponding residue with Gly-124 of B. sphaericus PheDH
is alanine in natural branched-chain amino acid dehy-
gtcatcgtc-3 . The DNA fragment encoded a ribosomal
binding site, an initiation codon, and the 6ꢀ His-tag
region derived from pRSET-B with additional EcoR I
0
0
and BamH I sticky ends at the 5 and 3 termini. It was
connected with pUC18 digested with EcoR I and
BamH I restriction enzymes. After sequence verifica-
tion, it was designated pUC18His, and was used as an
expression vector throughout this study. Wild-type
4
,5)
drogenases, LeuDH from Bacillus stearothermophilus
6
)
and ValDH from Streptomyces coelicolor. We have
constructed a single mutant of B. sphaericus PheDH, in
1
5,16)
17,18)
PheDH genes from B. badius
and B. sphaericus
7
)
which Gly-124 was replaced by Ala (Bs-G124A). This
substitution enhanced enzyme activity toward aliphatic
substrates as compared to the wild-type enzyme, but the
single mutant still showed the highest activity toward
L-Phe among the tested substrates, even though detailed
steady-state kinetic analysis showed significantly higher
specificity constants toward aliphatic substrates than
were subcloned into the BamH I and Pst I sites of
pUC18His, and these plasmids were named pBb and
pBs, respectively.
Single mutants of Bb-G123S from B. badius and of
Bs-G124S from B. sphaericus were constructed using a
QuikChange Site-directed Mutagenesis Kit (Stratagene,
La Jolla, CA). The following primers and complements
aromatic ones.8
)
were used: Bb-G123S, 5 -cgtttctatacaagtactgatatggg-3
and 5 -cccatatcagtacttgtatagaaacg-3 , and Bs-G124S,
0
0
0
0
Homocystinuria (HCU), caused by cystathionine
-synthase deficiency, is an important inborn error of
0
0
0
ꢁ
5 -cgattttacacaagtactgacatgggg-3 and 5 -ccccatgtcagtac-
0
metabolism leading to noticeable increases concentra-
tions of L-methionine in blood and of L-homocystine in
ttgtgtaaaatcg-3 . The products were transformed into
ꢁ
E. coli JM109. The transformants were grown at 37 C
for 12 h in LB broth, pH 7.5, supplemented with 50
9)
urine. In several countries, newborn screening for HCU
is carried out using blood specimens as well as the
mg/ml of ampicillin, and then 0.5 mM of isopropyl-ꢁ-D-
y
To whom correspondence should be addressed. Tel: +81-766-56-7500; Fax: +81-766-56-2498; E-mail: asano@pu-toyama.ac.jp

730
S. TACHIBANA et al.
Table 1. Relative Activities of Wild-Type and Mutant PheDHs with Aromatic and Aliphatic Substrates
Relative activity (%)
Substrates
B. badius
B. sphaericus
G124A7)
100
Wild-type14)
100
G123S
Wild-type16)
100
G124S
L-Phenylalanine
18
63
(1.09 U/mg)
b
b
b
b
b
(
71.6 U/mg)
(2.16 U/mg)
(114 U/mg)
72
(4.0 U/mg)
L-Tyrosine
L-Leucine
9.0
3.0
0.2
19
—a
100
89
7.8
57
89
87
52
80
67
—
23
67
1.3
0.45
3.9
1.4
1.3
3
L-Isoleucine
L-Norleucine
L-Valine
70
86
72
71
4.0
5.0
8.0
7.0
76
L-Norvaline
L-Methionine
L-Ethionine
64
23
100
98
6.4
3.1
39
Enzyme activity was measured at a concentration of 10 mM, except for L-tyrosine, at 4 mM.
aNo enzyme activity for L-tyrosine was detected.
bParentheses show specific activities.
thiogalactopyranoside was added to the medium and the
ꢁ
Table 2. Steady-State Kinetic Parameters of Wild-Type and Mutant
of B. badius PheDH
cells were further cultivated for 12 h at 30 C to induce
the enzyme protein.
Wild-type19)
G123S
Each cell paste was suspended in 5 times the volume
of 20 mM Tris–HCl buffer, pH 8.0, containing 0.3 M
NaCl, 5 mM 2-mercaptoethanol and 25 mM imidazole
Substrates
Km
kcat
ꢂ1
kcat=Km
Km
kcat
ꢂ1
kcat=Km
ꢂ1
ꢂ1
ꢂ1
ꢂ1
(
mM) (s ) (s mM
)
(mM) (s
)
(s mM
)
L-Phenylalanine 0.087 39
448
3.9
227
NDa NDa
1.55
0.0068
(
lysis buffer), and was disrupted by sonication for
ꢁ
L-Tyrosine
L-Leucine
8.6
ND
ND
ND
ND
ND
ND
34
NDa
0.47
0.39
0.20
0.16
0.17
0.058
20 min at 4 C with a Kubota Ultrasonicator Insonator
model 210 (19 kHz, Kubota, Tokyo). Cell debris was
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.3 1.54
3.8 1.48
8.1 1.60
10.7 1.67
8.6 1.47
9.7 0.559
L-Isoleucine
L-Norleucine
L-Valine
removed by centrifugation at 28;400 ꢀ g for 20 min at
ꢁ
4
C, and the supernatant was used as a cell-free extract.
The cell-free extract was applied to a Chelating-
Sepharose Fast Flow column (column volume, 10 ml)
equilibrated with lysis buffer. After a wash with 20 mM
Tris–HCl buffer, pH 8.0, containing 0.3 M NaCl, 5 mM
L-Norvaline
L-Methionine
aND, not determined.
2
-mercaptoethanol, and 75 mM imidazole, the adsorbed
enzyme was eluted with 20 mM Tris–HCl buffer, pH 8.0,
containing 0.3 M NaCl, 5 mM 2-mercaptoethanol, and
Substrate specificity toward various aromatic and
aliphatic amino acids of the single mutant enzymes was
examined. The relative activities are shown in Table 1.
Both single mutants, Bb-G123S and Bs-G124S, showed
high activity toward a wide variety of aliphatic
substrates. This tendency of Bb-G123S was more
prominent than that of the single mutants, G124A and
G124S, of B. sphaericus. The Bb-G123S mutant pref-
erentially oxidized the longer aliphatic branched-chain
amino acids (as for length, L-leucine > L-isoleucine >
L-valine and L-norleucine > L-norvaline, and as for
branching, L-leucine > L-isoleucine > L-norleucine, and
L-valine > L-norvaline), while the Bs-G124S mutant
preferentially oxidized the shorter straight-chain aliphat-
ic amino acids (as for length, L-valine > L-leucine >
L-isoleucine, and L-norvaline > L-norleucine, and as
for branching, L-norleucine > L-isoleucine > L-leucine,
and L-norvaline > L-valine).
To evaluate the substrate specificity in detail, we
examined the steady-state kinetic parameters on both
single mutants, as shown in Tables 2 and 3. A dramatic
decrease in the specific constant (kcat=Km value) of
Bb-G123S for L-Phe was caused by the increase in the
Km value for it (Table 2). On the other hand, the specific
constants for aliphatic substrates, except for L-Met,
appeared to depend on the side-chain length of their
substrates as well as the relative activity. No significant
difference in kcat values was observed between L-Phe
and aliphatic substrates, except for L-Met. Thus, a
difference in the substrate specificity of this mutant is
5
00 mM imidazole. The enzyme was dialyzed against
0 mM Tris–HCl buffer, pH 8.0, containing 0.1 mM
2
EDTA and 5 mM 2-mercaptoethanol overnight.
The oxidative deamination reaction, catalyzed by
mutants, was assayed by measuring the reduction of
þ
NAD at 340 nm in a 1 cm light path using a poly(meth-
ylmethacrylate) (PMMA) cuvette. The enzyme assay
1
7)
was performed as described previously. One unit of
the enzyme activity was defined as the amount of
enzyme that catalyzes the formation of 1 mmol of NADH
per min in oxidative deamination. The protein concen-
tration was determined with a Bio-Rad protein assay kit
(
2
Bio-Rad, Hercules, CA), or from the absorbance at
80 nm with bovine serum albumin as the standard.
Bb-G123S and Bs-G124S were purified to electro-
phoretically homogeneous as His-tag fusions. In both
cases, the molecular masses of the subunits were
estimated to be 43 kDa by SDS–PAGE, and were higher
than that of the original. The optimum temperature
ꢁ
ꢁ
(
65 C), thermal stability (55 C), and optimum pH
pH 10.4) of Bb-G123S were the same as those of the
(
wild-type from B. badius. On the other hand, these
1
5)
ꢁ
properties of Bs-G124S, optimum temperature (60 C),
ꢁ
thermal stability (50 C), and optimum pH (pH 10.4),
were slightly different from those of the wild-type from
B. sphaericus.1 Since these are relatively modest
changes as compared to the wild-type enzymes, they
have little impact on substrate specificities.
7)

Single Amino Acid Substitution of Phenylalanine Dehydrogenases
731
Table 3. Steady-State Kinetic Parameters of Wild-Type and Mutants of B. sphaericus PheDH
Wild-type8)
G124A8)
G124S
Substrates
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
Km (mM) kcat (s
)
kcat=Km (s mM
)
Km (mM) kcat (s
)
kcat=Km (s mM
)
Km (mM) kcat (s
)
kcat=Km (s mM
)
L-Phenylalanine
L-Tyrosine
1.1
1.3
68
68
61.8
52.3
1.3
8.1
ND
4.2
ND
1.0
1.1
1.6
0.52
0.11b
18
3.7
7.6
0.78
0.63
0.60
0.68
1.0
0.21
0.083
3.3
a
a
L-Leucine
0.55
0.14
0.79
2.0
0.70
0.057
0.11
0.096
0.13
0.19
0.73
0.18
0.19
0.45
0.29
0.26
0.45
L-Isoleucine
L-Norleucine
L-Valine
0.14
3.2
1.0
4.0
10
17
3.6
2.2
0.91
1.4
0.46
0.42
0.44
0.97
1.5
7.5
7.9
0.71
0.79
0.64
2.4
3.0
L-Norvaline
L-Methionine
3.3
2.7
1.2
1.4
1.9
1.4
aND, not determined.
bDetermined from the gradient of the linear part of the curve from a plot of activity against substrate concentration.
apparently determined by the substrate binding affinity
for each substrate. The specific constants for the
aromatic substrates, L-Phe and L-Tyr, in Bs-G124S also
dramatically decreased as compared to the wild-type of
B. sphaericus (Table 3). This alteration agrees with the
result for the single mutant Bs-G124A. In addition, the
kcat values for the aliphatic amino acids, including
L-Met, in each mutant, G124A and G124S, were
approximately of the same order. Nevertheless, the
specific constants for the aliphatic amino acids in
Bs-G124S showed only lower levels than those in
Bs-G124A. These results provide experimental evidence
that the enzyme activities and the specific constants
of the two mutants, Bb-G123S and Bs-G124S, have
broader specificity toward aliphatic substrates than those
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