Some properties of glycine aminotransferase purified from Rhodopseudomonas palustris No. 7 concerning extracellular porphyrin production.

Article abstract of DOI:10.1271/bbb.67.783

Glycine aminotransferase (EC 2.6.1.4; GlyAT) was presumed to be an enzyme concerning the supply of glycine for the extracellular porphyrin production by Rhodopseudomonas palustris No. 7. GlyAT was purified from strain No. 7 as an electrophoretically homogenous protein. The enzyme was a monomer protein with the molecular weight of about 42,000. From the absorption spectrum of the enzyme (350 nm, 410 nm), it was indicated that the enzyme had pyridoxal phosphate as a prosthetic group. The enzyme showed high substrate specificity for glutamate as an amino group donor. Apparent Kms for glutamate and glyoxylate were 6.20 mM and 3.75 mM, respectively. The Vmax and Kcat for glutamate were 66.8 mumol/min/mg protein and 46.8 s-1, respectively. The Vmax and Kcat for glyoxylate were 68.8 mumol/min/mg protein and 48.2 s-1. The optimum temperature and pH were 40-45 degrees C and 7.0-7.5, respectively. The enzyme activity lowered to about 50% in the presence of 15 mM ammonium chloride.

Full text of DOI:10.1271/bbb.67.783

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Biosci. Biotechnol. Biochem., 67 (4), 783–789, 2003
Some Properties of Glycine Aminotransferase Puriˆed
from Rhodopseudomonas palustris No. 7 Concerning
Extracellular Porphyrin Production
Hidetoshi YAMAGUCHI, Masahiro OHTANI, Seigo AMACHI
Hirofumi SHINOYAMA, and Takaaki FUJII
,
†
Department of Bioresources Science, Graduate School of Science and Technology, Chiba University,
648 Matsudo, Matsudo-shi, Chiba 271-8510, Japan
Received October 7, 2002; Accepted December 25, 2002
Glycine aminotransferase (EC 2.6.1.4; GlyAT) was
presumed to be an enzyme concerning the supply of
glycine for the extracellular porphyrin production by
Rhodopseudomonas palustris No. 7. GlyAT was
puriˆed from strain No. 7 as an electrophoretically
homogeneous protein. The enzyme was a monomer
protein with the molecular weight of about 42,000.
From the absorption spectrum of the enzyme (350 nm,
410 nm), it was indicated that the enzyme had pyridoxal
phosphate as a prosthetic group. The enzyme showed
high substrate speciˆcity for glutamate as an amino
in vivo have not been clariˆed.
Rhodopseudomonas palustris No. 7, isolated from
sewage, was excellent in the use of alcohols.5) When
strain No. 7 was growing in a medium containing
ethanol and malate as a carbon and hydrogen source,
these substrates were simultaneously consumed, and
the intracellular level of isocitrate lyase remarkably
rose.6,7) We found that this organism secreted por-
phyrin when it was incubated in the ethanol-malate
medium containing glutamate as a nitrogen source at
40
9
C. Porphyrin was hardly produced at 30
9
C.8,9)
group donor. Apparent
late were 6.20 m and 3.75 m
and Kcat for glutamate were 66.8
and 46.8 s－1, respectively. The
late were 68.8
optimum temperature and pH were 40
7.0 7.5, respectively. The enzyme activity lowered to
K
ms for glutamate and glyoxy-
In this paper, the glycine supply enzyme for the
M
M
, respectively. The Vmax
porphyrin production was searched for in R. palus-
tris No. 7, and the enzyme, glycine aminotransferase
(EC 2.6.1.4), was puriˆed as a single protein and its
characteristics were examined.
m
mol min mg protein
W
W
V
max and
K
cat for glyoxy-
m
mol min mg protein and 48.2 s－1. The
W
W
¿
459C and
¿
about 50
Materials and Methods
z
in the presence of 15 m
M
ammonium chlo-
ride.
Organism, medium, and growth conditions
.
Rhodopseudomonas sp. No. 7 and its maintenance
were described previously.5) Although the organism
had been tentatively positioned as Rhodopseudomo-
nas sp. No. 7, it was identiˆed as Rhodopseudomo-
nas palustris No. 7 afterwards.8,10) A basal salt
Key words: Rhodopseudomonas palustris; porphy-
rin; glycine aminotransferase; pho-
tosynthetic bacteria
It has been reported that purple non-sulfur bacte-
ria produced porphyrin extracellularly and the addi-
tion of glycine to the culture medium was eŠective
for the production.1–3) In these bacteria, porphyrin is
biosynthesized from glycine and succinyl-CoA in
passing through the Shemin pathway.4) Therefore,
the supply of intracellular glycine seems to be an im-
portant key to the extracellular production of por-
phyrin. Generally two routes are known as a glycine
supply system. One is the route that glycine is synthe-
sized from serine, and another is the route that gly-
cine is synthesized from glyoxylate by the transfer of
an amino group from amino acids. However, the de-
tails of the relation between the extracellular porphy-
rin production and the biosynthesis system of glycine
medium containing 0.1
bicarbonate, and 0.1 ammonium chloride was used
for the main culture.5) The organism was grown at
30 C in screw-capped 1.5-liter Roux bottles ˆlled
z ethanol, 0.2z sodium
z
9
with the medium. These bottles were illuminated at
an intensity of 3,000 lux with 60 W incandescent
lamps. After incubation, cells in the late logarithmic
growth phase were harvested by centrifugation. The
resultant cells were washed twice with 50 m
M
Tris
(hydroxymethyl) aminomethane (Tris)-HCl buŠer
－
(pH 8.0) and kept below 209C until use.
Extracellular production of porphyrin. For the
porphyrin production test, the cells cultivated in a
9
1.5-L Roux bottle at 30 C under light anaerobic con-
†
To whom correspondence should be addressed. Fax:
81-47-308-8866; E-mail: tfujii midori.h.chiba-u.ac.jp
＋ ＠

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784
H. YAMAGUCHI et al
.
ditions were used. The cells harvested in the
logarithmic growth phase were washed twice with
of NADH per min at 309C.
The activity of glutamate-oxaloacetate aminotrans-
ferase (EC 2.6.1.1) was examined by the formation
reaction of oxaloacetate from aspartate in the
presence of 2-oxoglutarate. The activity was assayed
by a coupled system in which the oxaloacetate
formed was reduced to malate by NADH in the
presence of malate dehydrogenase (Shiga).14) One
unit of an enzyme is deˆned as the amount of enzyme
50 m potassium phosphate buŠer (pH 7.0), and sus-
M
pended in various culture media for the test. The cell
suspension was illuminated at 10,000 lux in a 200-ml
Roux bottle equipment used for hydrogen genera-
tion.6,8) The initial cell concentration was adjusted to
an absorbance of about 1.0 at 660 nm. Growth and
porphyrin production were monitored by measuring
the optical density of the culture broth at 660 nm and
400 nm, respectively. Porphyrin was estimated as
coproporphyrin. The molar extinction coe‹cient
catalyzing the oxidation of 1
at 30 C.
mmol of NADH per min
9
3
－
1
×
・
of the coproporphyrin used was 7.47 10 (cm
Puriˆcation of glycine aminotransferase. For typi-
cal puriˆcation of GlyAT, the frozen cells (about
15 g, dry weight) obtained from 8.4 L of culture
M－1).11) The extracellular porphyrin was identiˆed by
thin-layer chromatography and high pressure liquid
chromatography as coproporphyrin.9)
broth were resuspended in 120 ml of 50 m
M
sodium
phosphate buŠer (pH 7.5), and then sonicated for
periods of 2 min in a 150 W Ohtake ultrasonic
disintegrator at 20 kHz. The total exposure time was
Enzyme assays. Cells harvested by centrifugation
were washed twice with 50 m
M
sodium-potassium
phosphate buŠer (pH 7.2), suspended in 10 ml of the
same buŠer to a concentration of about 0.1 g (wet
wt.) per ml, and then sonicated for periods of
2 min in a 150 W Ohtake ultrasonic disintegrator at
20 kHz. The total exposure time was 6 min. The son-
6 min. The sonic lysates were centrifuged at 25,000
×
g
for 20 min at 4 C. To the resultant supernatant, 2 g
9
of streptomycin sulfate was added to precipitate
nucleic acids. The precipitate was removed by cen-
trifugation, and the supernatant was fractionated by
ammonium sulfate at concentrations between 30 to
ic extracts were centrifuged at 10,000
at 4 C. The resultant supernatant was assayed for en-
zyme activity.
×
g for 20 min
9
70
dissolved in the Tris-HCl buŠer. The solution was
dialyzed against the 10 m the phosphate buŠer at
30 C overnight. The dialysate was put on a DEAE-
cellulose DE-52 (Whatman) column ( 2.6 40 cm)
z
saturation. The precipitate was collected and
The activity of glutamate-glyoxylate amino-
transferase, glycine aminotransferase, (EC 2.6.1.4;
GlyAT) was assayed principally according to the
M
9
q
×
method of Rowsell et al
tained an enzyme preparation, 18
(neutralized), 18 mol of sodium glutamate, and
30 mol of potassium phosphate buŠer (pH 7.5), in
0.6 ml total volume. The reaction was started by add-
ing glyoxylate at 30 C. After 20 min of incubation, it
was stopped by adding 0.6 ml of 3 HCl, and the
protein was removed by centrifugation. The resultant
sample was neutralized by 0.6 ml of 3 NaOH, and
.
12) The reaction mixture con-
equilibrated with the phosphate buŠer. The column
was washed with the buŠer, and eluted with a
1,000-ml linear gradient of sodium chloride (0 to
m
mol of glyoxylate
m
m
500 m ) dissolved in the same buŠer at a ‰ow rate of
M
20 ml h. Ten-ml fractions were collected. The active
W
9
fractions were combined and concentrated by an
ultraˆltration unit (Centriprep 30, Amicon). To the
concentrated solution, ammonium sulfate was added
N
N
to give 30
z saturation. The ammonium sulfate solu-
2-oxoglutarate in the sample was assayed by gluta-
mate dehydrogenase (Sigma) in the presence of
NADH and ammonium chloride. One unit of the en-
zyme was deˆned as the amount of enzyme catalyzing
tion was put on a butyl-Toyopearl 650M (Tosoh)
×
column (
buŠer containing 30
fate. The column was eluted with a 400-ml linear
q
1.6 40 cm) equilibrated with the Tris-HCl
z
saturation of ammonium sul-
the production of 1
m
mol of 2-oxoglutarate per min
gradient of ammonium sulfate (30 to 0z) dissolved
in the same buŠer at a ‰ow rate of 10 ml h. Four-ml
by monitoring NADH-oxidation.
W
The activity of aspartate-glyoxylate aminotrans-
ferase (EC 2.6.1.35) was assayed by a coupled system
in which the oxaloacetate formed was reduced to ma-
late by NADH in the presence of malate de-
hydrogenase (Sigma).13) One unit of an enzyme was
deˆned as the amount of enzyme catalyzing the oxi-
fractions were collected. The active fractions were
combined and concentrated by the ultraˆltration
unit. The concentrated solution was put on a Sepha-
×
(q1.6 100 cm)
dex G-100 (Pharmacia) column
equilibrated with the Tris-HCl buŠer. The column
was eluted with the same buŠer at a ‰ow rate of
dation of 1
m
mol of NADH per min at 30
9C.
10 ml h. Two-ml fractions were collected. The active
W
The activity of alanine-glyoxylate aminotrans-
ferase (EC 2.6.1.44) was assayed by a coupled system
in which the pyruvate formed was reduced to lactate
by NADH in the presence of lactate dehydrogenase
(Sigma).13) One unit of an enzyme is deˆned as the
fractions were combined and concentrated by the
ultraˆltration unit. The concentrated solution was
×
put on a Bio-Rad HTP (Bio-Rad) column (q1.6
100 cm) equilibrated with the Tris-HCl buŠer. The
column was eluted with the same buŠer at a ‰ow rate
amount of enzyme catalyzing the oxidation of 1
m
mol
of 10 ml h. Two-ml fractions were collected. The
W

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Properties of Glycine Aminotransferase Puriˆed from Rhodopseudomonas palustris
785
Table 1. EŠects of Addition of Amino Acids on Porphyrin
Production by Rhodopseudomonas palustris No. 7
active fractions were combined and concentrated.
－
The resultant preparation was stored at 209C.
Amino acid
(5 m
Porphyrin production
Protein assay. Protein was measured by the
method of Lowry et al
15) with bovine serum albumin
as a standard protein. Protein eluted from a column
was monitored by measuring the absorbance at
280 nm.
M
)
(mg L)
W
.
Glutamate
Glutamate glutamate
Glycine
29.5
31.7
6.96
40.2
5.67
26.9
0.24
4.63
＋
＋
Glutamate glycine
Serine
Glutamate serine
＋
Electrophoresis. The native protein sample was
NH4Cl
Glutamate NH4Cl
directly run on a 7.5
z
polyacrylamide gel in 50 m
M
＋
Tris-glycine buŠer (pH 8.3).16) Electrophoresis of the
denatured protein samples was done in 25 m Tris-
glycine buŠer (pH 8.7) containing 0.1 SDS on a
13
gel by the method of Laemmli.17)
In both cases, protein was stained by 0.25
Coomassie brilliant blue R-250 in 50 methanol
Various amino acids were added to the medium containing 0.1
and 0.1 malate. The cells were incubated at 40 C for 6 days.
z ethanol
M
z
9
z
z
z
that independently contained ethanol or malate,
respectively. As the nitrogen source, alanine and
aspartate were eŠective in the production except for
glutamate. Glycine is one of the starting materials in
porphyrin biosynthesis in photosynthetic bacteria.4)
In strain No. 7, its growth was not favorable in the
z
－
10
z
acetic acid solution.
Molecular weight estimation
.
The molecular
weight of the native enzyme was estimated by HPLC
on a TSK G3000 SW (Tosoh) column equilibrated
culture medium containing 5 m
M
glycine as the sole
with 50 m
M
potassium phosphate buŠer (pH 7.0).
source of nitrogen, and therefore, the production of
porphyrin was small in this case. Then, the sup-
plemental eŠect of glycine to the glutamate medium,
in which the production of porphyrin was good, was
examined (Table 1). The production was increased
The column was eluted with the same buŠer. Besides,
the molecular weight of the denatured enzyme was
estimated by SDS polyacrylamide gel electrophoresis
as described above.
around 35
z
by adding glycine. Strain No. 7 grew
Thin-layer chromatography. A silica gel plate with
lanes (Whatman silica gel 60) was used for identiˆca-
well in the medium containing serine as the nitrogen
source. Many organisms convert serine to glycine. In
the serine medium, however, the production of por-
phyrin was small, and the supplemental eŠect of ser-
ine to the glutamate medium was not observed. The
addition of ammonium chloride to the glutamate
medium greatly inhibited the production of porphy-
rin.
tion of amino acids. The plate was developed by 80
z
ethanol. Amino acids were detected by a ninhydrin
solution. For detection of glycine, an
hyde solution was sprayed.
o-phthalalde-
Measurement of UV and visible absorption spec-
tra. The UV and visible absorption spectra of the en-
zyme were measured by a Shimadzu recording spec-
trophotometer UV-160. The holoenzyme were meas-
Glycine synthesis from glyoxylic acid
Strain No. 7 simultaneously consumed ethanol and
malate when it was incubated in the ethanol-malate-
glutamate medium, and the glyoxylate cycle operated
at that time.7,18) Therefore, aminotransferase activity,
which used glyoxylate as an amino group acceptor
and glutamate, alanine, or aspartate as a donor,
was examined with the cell-free extracts prepared
from strain No. 7 grown in the production medium
(Table 2)‚
ured in 10 m
M
potassium phosphate buŠer solution
(pH 7.0). The holoenzyme of the pyridoxamine type
was prepared by incubating with the same buŠer
solution including 30 m
30 C. The apoenzyme was prepared by dialyzing
with 500 m
M
glutamate for 30 min at
9
M
potassium phosphate buŠer solution
(pH 7.0) including 30 m
M
glutamate.
The activity using aspartate as the donor hardly
could be detected. In all extracts examined, though
the activities using alanine and glutamate were
found, glutamate gave an activity that was higher
than alanine about 3 times. The activity of serine-
glycine aminotransferase was not otherwise detected
(data not shown). These results indicated that the
glutamate-dependent enzyme played the main role
for the synthesis of glycine from glyoxylate in R.
palustris No. 7. It was estimated that this enzyme
corresponded to the systematic name of glycine: 2-
Results
EŠects of amino acids on the extracellular produc-
tion of porphyrin
The extracellular production of porphyrin (about
35 mg L) by R. palustris No. 7 was observed, when it
W
was cultivated in the basal salt medium containing
ethanol and malate as an electron-carbon source
9
and glutamate as a nitrogen source at 40 C. But the
production of porphyrin was greatly lowered when
the bacterium was cultivated in the culture medium

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786
H. YAMAGUCHI et al
.
oxoglutarate aminotransferase (EC 2.6.1.4; GlyAT).
The enzymatic speciˆc activity of this GlyAT was in-
creased after the growth of strain No. 7 entered in the
logarithmic phase. The activity was greatest after
about 72 h and then gradually decreased in the sta-
tionary growth phase.
Molecular weight and subunit structure
In polyacrylamide electrophoresis (SDS-PAGE),
the molecular weight of GlyAT was estimated to be
about 42,000 as a result of comparing the standard
protein with the mobility of GlyAT. There was
Puriˆcation of GlyAT from R. palustris No. 7
GlyAT of R. palustris No. 7 was puriˆed from cells
grown in the medium containing 0.2
z
ethanol and
0.2
z bicarbonate. A typical puriˆcation of GlyAT is
summarized in Table 3.
In strain No. 7, the activity of glutamate-oxalo-
acetate aminotransferase (GOT) was considerably
stronger than the activity of other aminotransferases.
In spite of that, it was possible to easily separate the
GlyAT-fraction from the GOT-fraction by the
DEAE-cellulose column chromatography (Fig. 1).
The activity using glyoxylate as the acceptor was not
found in the GOT-fraction. The puriˆed enzyme
Fig. 1. Chromatography of GlyAT from Rhodopseudomonas
palustris No. 7 on DEAE Cellulose DE 52.
×
M
F2.6 40 cm) was eluted with 50 m potassium
The column (
phosphate buŠer (pH 7.5) containing sodium chloride (0–
(0.753 mg) was obtained in a yield of 8.31
z
without
a special stabilizer and had the speciˆc activity of
#
500 m ). ( ), GlyAT activity; ( ), GOT activity; ( ), absor-
M
 
bance at 280 nm.
3.25 U mg protein. The ˆnal puriˆcation factor was
W
about 190-fold. The activity of the puriˆed enzyme
showed no great loss during storage over eight
－
9
months at 20 C.
The puriˆed enzyme was homogeneous as judged
by the criterion of non-denaturing polyacrylamide
gel electrophoresis (Fig. 2(A)). Moreover, no
evidence of contamination was found on SDS-
polyacrylamide gel electrophoresis (Fig. 2(B)).
Table 2. EŠects of Carbon Sources in Culture Medium on Activ-
ity Levels of Glyoxylate Aminotransferase in Rhodopseudomonas
palustris No. 7
Enzyme activity of aminotransferase
Carbon source
(nmol min mg protein)
W W
(0.1
z
)
Asp-GAT
Ala-GAT
Glu-GAT
Fig. 2. Polyacrylamide Gel Electrophoresis of GlyAT from
Rhodopseudomonas palustris No. 7.
Ethanol
Malate
Ethanol
º
º
º
1
1
1
2.53
2.61
3.41
9.89
8.44
10.1
A: polyacrylamide gel (7.5
z
). B: SDS-polyacrylamide gel
＋
malate
(13 ). Lane 1, GlyAT. Lane 2, Mr standards: phosphorylase b
z
(94,000), bovine serum albumin (67,000), ovalbumin (43,000),
carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100),
Asp-GAT, Aspartate-glyoxylate aminotransferase. Ala-GAT, Alanine-
glyoxylate aminotransferase. Glu-GAT, Glutamate-glyoxylate aminotrans-
ferase (GlyAT). The cells were incubated in the medium containing various
a
-lactalbumin (14,400). Lane 3, GlyAT not treated with b-mer-
captoethanol. Lane 4 GlyAT treated with
b
-mercaptoethanol.
carbon sources and 0.1
z ammonium chloride at 309C.
Table 3. Summary of Puriˆcation of GlyAT from Rhodopseudomonas palustris No. 7
Total enzyme
activity
Speciˆc
activity
Volume
(ml)
Protein
(mg)
Yield
Puriˆcation
(fold)
Puriˆcation procedure
(
z)
(U)
(U mg)
W
Cell-free extract
Streptomycin treatment
114
112
42
86
5.1
16
1740
1530
1070
18.2
3.85
2.55
0.753
29.5
25.9
20.7
10.2
4.82
4.30
2.45
0.0170
0.0170
0.0193
0.560
1.25
1.69
3.25
100
1.00
1.00
1.12
32.9
73.5
99.4
191
87.8
70.2
34.6
16.3
14.6
30–70
z (NH4)2SO4 sat.
DEAE Cellulose DE52
BUTYL-TOYOPEARL
Sephadex G-100
Hydroxyapatite
6.5
8.31