Purification of three aminotransferases from
Hydrogenobacter thermophilus TK-6 – novel types of
alanine or glycine aminotransferase
Enzymes and catalysis
Masafumi Kameya, Hiroyuki Arai, Masaharu Ishii and Yasuo Igarashi
Department of Biotechnology, The University of Tokyo, Japan
Introduction
Aminotransferase (EC 2.6.1) catalyses the conversion
between amino acids and 2-oxo acids, transferring the
amino group of the amino acid onto the 2-oxo acid.
This enzyme is widespread, being present in almost
all organisms, and plays a key role in the synthesis
and degradation of amino acids. As the substrates ⁄
products of aminotransferase, namely 2-oxo acids and
amino acids, are key metabolites in carbon and nitro-
gen metabolism, this enzyme can be regarded as a
physiologically important linkage within central meta-
bolism. Furthermore, some aminotransferases have
been reported to be coupled with further metabolic
Keywords
2-oxo acid; amino acid; aminotransferase;
Hydrogenobacter thermophilus; nitrogen
anabolism
Correspondence
M. Ishii, Department of Biotechnology,
The University of Tokyo, Yayoi 1-1-1,
Bunkyo-ku, Tokyo 113-8657, Japan
Fax: +81 3 5841 5272
Tel: +81 3 5841 5143
E-mail: amishii@mail.ecc.u-tokyo.ac.jp
(Received 6 January 2010, revised 27
January 2010, accepted 2 February
2010)
doi:10.1111/j.1742-4658.2010.07604.x
Aminotransferases catalyse synthetic and degradative reactions of amino
acids, and serve as a key linkage between central carbon and nitrogen
metabolism in most organisms. In this study, three aminotransferases (AT1,
AT2 and AT3) were purified and characterized from Hydrogenobacter
thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium, which
has been reported to possess unique features in its carbon and nitrogen
anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate amino-
transferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate
aminotransferase activities, respectively. In addition, both AT1 and AT2
catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly,
phylogenetic analysis showed that AT2 belongs to aminotransferase
family IV, whereas known glutamate:pyruvate aminotransferases and gluta-
mate:glyoxylate aminotransferases are members of family Ic. In contrast,
AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate
aminotransferases which belong to family IV. Although Thermococcus
litoralis alanine:glyoxylate aminotransferase is the sole known example of
family I alanine:glyoxylate aminotransferases, it is indicated that this
alanine:glyoxylate aminotransferase and AT3 are derived from distinct lin-
eages within family I, because neither high sequence similarity nor putative
substrate-binding residues are shared by these two enzymes. To our knowl-
edge, this study is the first report of the primary structure of bacterial gluta-
mate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase,
and demonstrates the presence of novel types of aminotransferase phyloge-
netically distinct from known eukaryotic and archaeal isozymes.
Abbreviations
AGT, alanine:glyoxylate aminotransferase; CFE, cell-free extract; GGT, glutamate:glyoxylate aminotransferase; GOT, glutamate:oxaloacetate
aminotransferase; GPT, glutamate:pyruvate aminotransferase; 2-OG, 2-oxoglutarate; PLP, pyridoxal 5¢-phosphate; PSOT, phosphoserine:
2-oxoglutarate aminotransferase.
1876 FEBS Journal 277 (2010) 1876–1885 ª2010 The Authors Journal compilation ª2010 FEBS
activities, e.g. enzymes involved in the malate shuttle,
porphyrin synthesis [1], maintenance of intracellular
redox status [2] or plant photorespiration [3].
A wide variety of substrates for aminotransferases
have been reported, including branched-chain amino
acids, aromatic amino acids, b-amino acids and their
corresponding 2-oxo acids. To categorize diverse amin-
otransferases, classifications based on the primary
structure have been proposed. Such a classification
divides aminotransferases into four families, numbered
I–IV [4]. Family I is further divided into several
subfamilies, such as Iaand Ic[5]. In this classification
system, enzymes belonging to the same family or
subfamily share common enzymatic characteristics to
some extent.
However, the substrate specificities of aminotransfe-
rases are diverse, even within the same family or sub-
family; therefore, at present, it is difficult to predict
the specificities on the basis of the primary structures
only. One reason for this difficulty is that the reaction
mechanisms and structures of aminotransferases may
be similar to each other, even if they react specifically
with different substrates. Moreover, there are only a
limited number of aminotransferases whose enzymatic
properties and primary sequences have been deter-
mined. For these reasons, the function of most puta-
tive aminotransferase homologues found in the
genome database remains to be ascertained. Some
recent studies have revealed properties of several puta-
tive aminotransferases by biochemical and enzymatic
analyses [6–8], demonstrating the importance of a bio-
chemical approach for the characterization of these
enzymes.
Hydrogenobacter thermophilus TK-6 is a thermo-
philic, hydrogen-oxidizing, obligately chemolithoauto-
trophic bacterium. The analysis of 16S rRNA
sequences has shown that Hydrogenobacter species are
located on the deepest branch in the domain Bacteria
on the phylogenetic tree, together with other Aquificae
species [9]. Reflecting this distinctive phylogenetic posi-
tion, this bacterium shows many unique characteristics.
One such characteristic is its carbon anabolism, where
carbon dioxide is fixed via the reductive tricarboxylic
acid cycle. Key enzymes in this cycle have been charac-
terized and shown to have novel enzymatic features
[10–13]. Furthermore, enzymatically peculiar character-
istics have also been found in this bacterium’s nitrogen
anabolism [14,15]. Although previous studies have
demonstrated that H. thermophilus assimilates nitrogen
in the form of ammonium to produce glutamate (Glu),
it has not yet been clarified how Glu serves as the
nitrogen donor for the synthesis of other nitrogenous
compounds.
The study of aminotransferases in this bacterium is
of interest, firstly because of the need to characterize
biochemically aminotransferases. The importance of
this is emphasized by the belief that a novel amino-
transferase would be found in this phylogenetically
deep-rooted bacterium. Secondly, this study was
expected to lead to further elucidation of the metabo-
lism of H. thermophilus. Such elucidation would not be
restricted to nitrogen metabolism, but would also
include its unique central carbon metabolism. In this
study, three aminotransferases were purified and
characterized biochemically and presumed to contrib-
ute to aspartate (Asp), alanine (Ala) and glycine (Gly)
syntheses. Phylogenetic analysis of these enzymes
showed a unique combination of substrate specificities
and phylogenetic positions, providing novel insights
into the aminotransferase classification.
Results
Aminotransferase activities in cell-free extract
(CFE)
Given that H. thermophilus operates a distinctive carbon
pathway, the reductive tricarboxylic acid cycle, its central
carbon metabolism is of interest. Therefore, we focused
on amino acids with relatively simple carbon skeletons:
Glu, Asp, Ala and Gly. Aminotransferase activities in the
CFE were assayed combining Glu, Asp, Ala or Gly as the
amino group donor and 2-oxoglutarate (2-OG), oxaloac-
etate, pyruvate or glyoxylate as the amino group accep-
tor. Consequently, the following four kinds of activity
were detected: 0.96 UÆmg
)1
glutamate:oxaloacetate
aminotransferase (GOT; EC 2.6.1.1), 0.30 UÆmg
)1
gluta-
mate:pyruvate aminotransferase (GPT; EC 2.6.1.2),
0.30 UÆmg
)1
glutamate:glyoxylate aminotransferase
(GGT; EC 2.6.1.4) and 0.07 UÆmg
)1
alanine:glyoxylate
aminotransferase (AGT; EC 2.6.1.44). Although the
GOT reaction was catalysed reversibly, the other
reactions proceeded irreversibly as follows:
GOT: Glu + oxaloacetate $2-OG + Asp
GPT: Glu + pyruvate !2-OG + Ala
GGT: Glu + glyoxylate !2-OG + Gly
AGT: Ala + glyoxylate !pyruvate + Gly
Although GOT is a representative aminotransferase
that has been studied extensively in many organisms
[16–18], other aminotransferases have been less well
studied, especially in bacteria. GPT has been purified
and characterized in a few organisms, and only a
M. Kameya et al. Three aminotransferases from H. thermophilus
FEBS Journal 277 (2010) 1876–1885 ª2010 The Authors Journal compilation ª2010 FEBS 1877
limited number of GPT sequences have been deter-
mined [2,6,19]. GGT and AGT have been subjected to
considerably less research. GGT has been purified
from a few organisms [20], and only those from
Arabidopsis thaliana have been sequenced [3]. AGT has
been sequenced and characterized in eukaryotes and
archaea [21,22], but not in bacteria. Because of this
background, the characterization of these aminotrans-
ferase activities was expected to provide new insights
into bacterial aminotransferases.
Purification and phylogenetic analysis of
aminotransferases
Enzymes that exhibited GOT, GPT, GGT or AGT
activity were subjected to purification, and three
enzymes (AT1, AT2 and AT3) were purified from
H. thermophilus CFE (Table 1). It was shown that
GOT, GPT and AGT activities were derived from the
single enzymes AT1, AT2 and AT3, respectively
(Fig. 1). GGT activity was caused by AT1 and AT2,
which exhibited 11 and 60 UÆ(mg purified protein)
)1
of
GGT activity, respectively. No other enzymes that
exhibited GOT, GGT, GPT or AGT activity were
detected throughout the purification, suggesting that
the four kinds of activity in CFE were derived from
only the three enzymes. Purified AT1, AT2 and AT3
gave single bands of 44, 42 and 45 kDa on SDS
⁄PAGE, respectively (Fig. 2). The N-terminal amino
acid sequences of AT1, AT2 and AT3 were determined
to be MNLSKRVSHIKPAPT, MYQERLFTPG and
MSEEWMFPKVKKL, respectively, and the full-
length genes were identified in the H. thermophilus
genome (AP011112). The molecular masses of AT1,
AT2 and AT3 were calculated from their deduced pro-
tein sequences to be 43.7, 41.9 and 45.6 kDa, respec-
tively. These masses were consistent with those
calculated from SDS ⁄PAGE.
The phylogenetic tree was constructed on the basis
of the amino acid sequences (Fig. 3). GOT is known
to be divided into two groups in subfamilies Iaand Ic,
and AT1 belongs to aminotransferase subfamily Ic
together with some other GOTs. Unexpectedly, AT2 is
classified into family IV together with eukaryotic
peroxisomal AGT, whereas other GPTs are members
of family I. Interestingly, AT3 was located in family I,
unlike eukaryotic AGT. There is only one report of a
family I AGT, which was purified from Thermococ-
cus litoralis [22]. The order of divergence of AT3 from
enzymes in subfamily Icis ambiguous in Fig. 3
Table 1. Purification of AT1, AT2 and AT3 from H. thermophilus.
Enzyme Fraction
Activity
(U)
a
Protein
(mg)
Specific activity
(UÆmg
)1
)
a
Purification
(fold)
Yield
(%)
AT1 CFE 636 660 0.96 1 100
Butyl-Toyopearl 245 13 19 20 39
DEAE-Toyopearl 74 1.3 59 61 12
MonoQ 61 0.26 239 248 10
AT2 CFE 275 927 0.30 1 100
Butyl-Toyopearl 65 24 2.7 9 24
DEAE-Toyopearl 31 3.0 10 35 11
Hydroxyapatite 15 0.3 51 171 5
MonoQ 10 0.13 79 266 4
AT3 CFE 129 1811 0.071 1 100
Butyl-Toyopearl 14 71 0.19 3 11
DEAE-Toyopearl 5.4 3.7 1.4 20 4
Hydroxyapatite 2.1 0.42 5.0 69 2
MonoQ 2.9 0.36 8.0 112 2
Phenyl Superose 1.2 0.063 19 270 1
a
Representing GOT activity (in the direction of Asp synthesis) for AT1, GPT activity for AT2 and AGT activity for AT3.
Asp
Glu
2-OG
OAA
AT1
2-OG
Pyr
Ala
AT2
2-OG
Glyo
Gly
AT2 & AT1
PyrGlyo
AT3
Fig. 1. Aminotransferase reactions catalysed by AT1, AT2 and AT3.
Glyo, glyoxylate; OAA, oxaloacetate; Pyr, pyruvate.
Three aminotransferases from H. thermophilus M. Kameya et al.
1878 FEBS Journal 277 (2010) 1876–1885 ª2010 The Authors Journal compilation ª2010 FEBS
because of the low bootstrap values, although more
detailed phylogenetic analysis indicated that AT3 is
positioned separately from the known members of
subfamily Ic(see below).
Enzymatic properties
Gel filtration estimated the molecular mass of AT1 to
be 78 kDa, indicating that this enzyme forms a dimer
of two identical subunits, as do many known amin-
otransferases. The molecular masses of AT2 and AT3
were estimated to be 62 and 69 kDa, respectively.
These values were 1.5-fold larger than each single
peptide mass, indicating that these enzymes are mono-
mers or homodimers. Considering that some thermo-
philic enzymes have compact folding and their
molecular masses are often underestimated by gel
filtration [14], AT2 and AT3 might form a homodimer,
although it cannot be excluded that they are mono-
meric.
The effects of pH on the aminotransferase activities
of AT1, AT2 and AT3 were tested. AT1 exhibited the
highest GOT activities in both directions over a broad
pH range, 6.9–7.9 at 70 C. AT2 and AT3 showed the
highest GGT and AGT activities, respectively, at
pH 7.9–8.4. These natural or slightly basic optimum
pH values are common among known aminotransfe-
rases. Some aminotransferases are known to be acti-
vated by the addition of pyridoxal 5¢-phosphate (PLP),
the catalytic cofactor of aminotransferase, to the reac-
tion mixture [2]. The addition of PLP did not affect
the activities of AT1, AT2 or AT3, suggesting that
PLP binds tightly to these enzymes or extrinsic PLP
cannot reactivate the apoenzymes.
AT1 catalyses the GOT reaction reversibly and the
GGT reaction only in the direction of Gly synthesis.
AT2 catalyses the GPT reaction in the direction of Ala
synthesis, and shows only trace activity (< 5% of that
in the forward direction) in the reverse direction. This
enzyme also irreversibly catalyses the GGT reaction in
the direction of Gly synthesis, as well as AT1. Many
known GPTs catalyse the GPT reaction reversibly and
lack GGT activity. GPTs from A. thaliana share these
properties with AT2 [3], although these GPTs belong
to subfamily Icdistant from AT2, which is a member
of family IV (Fig. 3). AT3 specifically catalyses the
AGT reaction irreversibly in the direction of Gly
synthesis. The irreversibility of GGT and AGT is a
common feature among known GGTs and AGTs
[20,22,23]. Although some eukaryotic AGTs have been
reported to exhibit serine:pyruvate aminotransferase
activity [21], AT3 did not show this activity, suggesting
a high substrate specificity for Ala and glyoxylate
compared with these AGTs.
Some members of family IV are known as phospho-
serine:2-oxoglutarate aminotransferases (PSOT;
EC 2.6.1.52), which catalyse the conversion of phos-
phoserine and 2-OG to phosphohydroxypyruvate and
Glu [7,24,25]. AT2, which belongs to family IV, exhib-
ited PSOT activity at 16 UÆmg
)1
, corresponding to
about one-quarter of its GGT activity. It is noteworthy
that, although AT2 has a higher similarity to known
AGTs than to known PSOTs, it does not have AGT
activity but shows PSOT activity (Fig. 3).
Kinetic characterization
The kinetic parameters of AT1, AT2 and AT3 were
determined for the reactions that followed typical
Michaelis–Menten kinetics (Table 2). AT1 exhibited
higher V
max
values in GOT reactions than in the GGT
reaction. K
m
values for Glu, Asp and 2-OG in the
GOT reaction were comparable with those of other
reported GOTs [16,26]. With regard to GGT activity,
both AT1 and AT2 showed K
m
values as low as those
of known GGTs [3,20]. Although the GGT specific
activity of AT1 was less than one-fifth of that of AT2,
both specific activities were higher than those of
reported GGTs (such as 5.71 UÆmg
)1
from A. thaliana
and 3.25 UÆmg
)1
from Rhodopseudomonas palustris).
These data indicate that, not only AT2, but also AT1
has GGT catalytic efficiency comparable with or
12 3 4
(kDa)
97
66
45
31
22
14
Fig. 2. SDS ⁄PAGE (13%) of purified AT1, AT2 and AT3. Lane 1,
purified AT1; lane 2, purified AT2; lane 3, purified AT3; lane 4,
molecular mass markers.
M. Kameya et al. Three aminotransferases from H. thermophilus
FEBS Journal 277 (2010) 1876–1885 ª2010 The Authors Journal compilation ª2010 FEBS 1879
higher than that of known enzymes. AT2 also showed
GPT activity, but its K
m
value for pyruvate was too
high to determine accurately. Further investigations
are required to verify the extent to which AT2 contrib-
utes to the GPT reaction in vivo.K
m
values of AT3
were estimated to be equivalent to those of known
AGTs.
All determined K
m
values, except for that of AT2
for pyruvate, were less than or equivalent to those of
known aminotransferases. These results indicate that
AT1, AT2 and AT3 are adequately efficient to serve as
GOT or GGT, GGT or PSOT, and AGT, respectively.
Discussion
In this study, GOT, GGT, GPT and AGT activities
were detected in H. thermophilus, and three
aminotransferases were identified. These activities are
believed to enable this bacterium to synthesize Asp,
Ala and Gly by transferring the amino group of Glu
as the nitrogen source. These enzymes were completely
purified and characterized and, as such, this report
represents, to our knowledge, the first description of
the characterization of bacterial GGT and AGT at an
enzymatic and gene level.
Comparison of the amino acid sequences with
known enzymes showed the phylogenetic position of
each aminotransferase. AT2 showed high similarity to
eukaryotic AGT in family IV, whereas AT2 possessed
GGT, GPT and PSOT activities instead of AGT
activity. Most GGTs have been reported to lack GPT
activity, with the exception of the GGT from
A. thaliana [3]. In addition, GPTs have been identified
in several organisms, such as Corynebacterium glutami-
cum,Pyrococcus furiosus and mammals [2,6,19], and
all are classified into subfamily Icrather than into
family IV. Therefore, it is obvious that AT2 is phylo-
genetically distinct from known GGTs and GPTs. AT2
also possessed PSOT activity, which is found in some
enzymes belonging to family IV. A study of the struc-
ture of the Escherichia coli PSOT identified several
conserved residues that bind to the substrates [25].
His41, Arg42, His328 and Arg329 in the E. coli PSOT
are involved in the interaction with the negatively
charged phosphate group of the phosphoserine. These
residues are conserved not in AGTs, but are found in
all PSOTs (Fig. S1, see Supporting information). Inter-
estingly, AT2 harbours two of these four conserved
residues (His29 and Arg30 in AT2). It may be that
these partially conserved residues endow AT2 with
PSOT activity, which is uncommon among known
AGTs of family IV.
AT3 also occupies an unusual phylogenetic position
in family I, considering that this enzyme exhibited
Fig. 3. Phylogenetic tree of aminotransfe-
rases on the basis of the amino acid
sequences. The numbers at the nodes are
bootstrap confidence values expressed as
percentages of 1000 bootstrap replicates.
The order of the divergence was presumed
to be reliable only when the bootstrap
values were above 50. The tree was con-
structed using the neighbor-joining method
and showed the same overall topology as
that constructed by the maximum likelihood
method. Plus signs indicate the activities
proven experimentally. The accession num-
bers of each enzyme are shown in paren-
theses. Enzymes from the following
organisms were used: Arabidopsis thaliana
[3,21,26], Bacillus circulans [24], Bacillus sp.
YM-2 [17], Corynebacterium glutamicum [6],
Escherichia coli [25], Entamoeba histolytica
[7], human [35], H. thermophilus,
Pyrococcus furiosus [2,36], rat [19,37,38],
Saccharomyces cerevisiae [39], Sulfolobus
solfataricus [40], T. litoralis [22] and
Thermus thermophilus [18].
Three aminotransferases from H. thermophilus M. Kameya et al.
1880 FEBS Journal 277 (2010) 1876–1885 ª2010 The Authors Journal compilation ª2010 FEBS