Hydrogenobacter thermophilus TK-6 – novel types ofalanine or glycine aminotransferase Enzymes and catalysis Masafumi Kameya, Hiroyuki Arai, Masaharu Ishii and Yasuo Igarashi Department o
Trang 1Hydrogenobacter 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.
Trang 2activities, 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 Ia and 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)1alanine: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
Trang 3limited 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 thalianahave 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))1of
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 Ia and 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 Ic is ambiguous in Fig 3
Table 1 Purification of AT1, AT2 and AT3 from H thermophilus.
Activity (U) a
Protein (mg)
Specific activity (UÆmg)1) a
Purification (fold)
Yield (%)
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
Pyr Glyo
AT3 Fig 1 Aminotransferase reactions catalysed by AT1, AT2 and AT3 Glyo, glyoxylate; OAA, oxaloacetate; Pyr, pyruvate.
Trang 4because 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 homodimono-mers 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 70C 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 addireac-tion 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 Ic distant 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 Vmaxvalues in GOT reactions than in the GGT reaction Km 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 Kmvalues 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
(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.
Trang 5higher than that of known enzymes AT2 also showed
GPT activity, but its Km 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 Km values of AT3
were estimated to be equivalent to those of known
AGTs
All determined Km 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 Ic rather 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].
Trang 6AGT activity An AGT belonging to family I has only
been found in T litoralis [22] This AGT has several
characteristics similar to those of AT3, such as
compa-rable specific activity (29 UÆmg)1) and strict substrate
specificity However, AT3 seems to be phylogenetically
distant from the T litoralis AGT, because of the low
similarity between them: AT3 shows 26% identity to
AGT, which is lower than the identity between AT3
and Thermus thermophilus GOT (31%) Furthermore,
AT3 lacks several residues that are presumed to affect
the substrate specificity of the T litoralis AGT, e.g
Thr108 in the T litoralis AGT is supposed to serve to
the specificity for Ala [27], but this residue is replaced
by Lys105 in AT3 (Fig S2, see Supporting
informa-tion) In addition, Leu19, which is located near the
substrate in T litoralis AGT, is replaced by Phe18 in
AT3 These phylogenetic and structural differences
suggest that AT3 has a substrate recognition
mecha-nism distinct from that presumed in the T litoralis
AGT
The high similarity between the T litoralis AGT and
kynurenine aminotransferase II [28,29] has noted [27],
and they also share similarity with a-aminoadipate
aminotransferase [30] and aromatic aminotransferase
[31] These enzymes form a cluster in the phylogenetic
tree, but AT3 is clearly located outside of the cluster
(Fig 4) This position also supports the phylogenetic
dissimilarity between AT3 and T litoralis AGT
Instead of these enzymes, AT3-like genes are found in
genomes of Aquificales and c- or d-proteobacteria
(a few of the homologues are depicted in Fig 4) None
of these homologues has been subjected to biochemical studies, and their enzymatic properties and functions are of interest
It has been shown that H thermophilus has GGT activity and that this activity is derived from two enzymes, AT1 and AT2, with specific activities signifi-cantly higher than those of known GGTs GGT activi-ties derived from AT1 and AT2 in the CFE are calculated to be 0.044 and 0.23 UÆmg)1, respectively, from the specific activities and purification factors of each enzyme These values indicate that most of the GGT activity can be attributed to AT2 Although functional analyses for aminotransferase in vivo are necessary to clarify their physiological roles, it can be speculated that AT2 plays a major role in the GGT reaction to synthesize Gly, and AT1 mainly serves in the GOT reaction
Although no bacterial GGT gene has been identified, GGT purification has been reported from two species, Rhodopseudomonas palustris and Lacto-bacillus plantarum [20,23] AT1 and AT2 homo-logue genes are found in the genomes of both species (NP_949667 and NP_946142 in R palustris; NP_785312 and NP_784469 in L plantarum), and it is possible that the reported GGT activities were derived from these gene products Further biochemical research is needed to clarify the distribution of these types of homologue with GGT activity
One of the noteworthy findings in this study is that AT2 and AT3 showed novel substrate specificities from the viewpoint of the well-established aminotransferase classification (Fig 3), suggesting that the substrate specificity of aminotransferases is broader than previ-ously known The enzymatic data obtained are expected to be of use in predicting the function of putative aminotransferase homologues that are found
in the genome database It remains unclear whether similar aminotransferases are distributed among a broad range of organisms or whether these enzymes evolved after the divergence from other bacteria early
in evolution Further biochemical study is needed to solve this question Another intriguing question con-cerns glyoxylate metabolism in H thermophilus Although all three aminotransferases purified in this work use glyoxylate as their substrate, no enzymatic activities for the glyoxylate cycle were detected (not shown), and no genes encoding these enzymes are found in the genome Glycolate oxidase (EC 1.1.3.15), which catalyses the conversion of glycolate into glyoxylate, may be one of the candidates for physio-logical glyoxylate synthesis Several genes in the
H thermophilus genome share similarity with those of
Table 2 Kinetic parameters of AT1, AT2 and AT3 (ND, not
deter-mined).
Enzyme Reaction Substrate Km(m M )
Apparent
Vmax (UÆmg)1)
Oxaloacetate a 0.38 ± 0.05 240 ± 10
PSOT Phosphoserine 0.66 ± 0.07 17 ± 0
Glyoxylate 0.90 ± 0.08 24 ± 1 a
The estimate of the K m value for oxaloacetate may be higher than
the true value because of the instability of oxaloacetate at the
assay temperature.
Trang 7glycolate oxidase However, it remains unclear whether
these genes actually encode glycolate oxidase and,
fur-thermore, no genes have been found to explain how
glycolate can be synthesized in this bacterium
More-over, elucidation of an unidentified carbon metabolism
is needed to explain glyoxylate and Gly biosyntheses in
this bacterium Studies to clarify these pathways are in
progress, and these may elucidate a novel central
carbon metabolism in this bacterium
Materials and methods
Bacterial strain and growth conditions
Hydrogenobacter thermophilus TK-6 (IAM 12695, DSM
6534) was cultivated in an inorganic medium at 70C
under a gas phase of 75% H2, 10% O2and 15% CO2, as
described previously [32] Ammonium sulfate in the
med-ium and CO2 in the gas phase were the sole nitrogen and
carbon sources, respectively
Aminotransferase assay
Reaction mixtures contained 50 mm NaPO4(pH 8.0), 5 mm
amino acid, 5 mm 2-oxo acid and the enzyme solution If
necessary, 100 lm PLP was added For GOT, GGT, GPT,
AGT and PSOT assays, substrate concentrations were
mod-ified as follows: 100 mm Glu and 10 mm oxaloacetate or
10 mm Asp and 10 mm 2-OG for GOT, 20 mm Glu and
20 mm glyoxylate for GGT, 20 mm Glu and 30 mm pyru-vate for GPT, 40 mm Ala and 5 mm glyoxylate for AGT, and 10 mm phosphoserine and 10 mm 2-OG for PSOT For the AT1 assay, the pH in the reaction mixture was changed
to 7.2 The reaction mixtures were incubated at 70C, the optimum growth temperature of this bacterium Amino-transferase activities were determined by measuring the production of the amino acid or the 2-oxo acid
To measure amino acid production, the reaction mix-tures were subjected to phenylthiocarbamyl derivatization, and the derivatized samples were analysed with a reverse-phase column (Inertsil ODS-3, 4.6 mm· 25 cm; GL Science, Tokyo, Japan) to determine the amino acid production [14] One unit of activity was defined as the activity producing 1 lmol of an amino acid or a 2-oxo acid per minute
To measure 2-oxo acid production, 150 lL of the reac-tion mixtures were incubated at 70C and the reaction was stopped by the addition of 16 lL of 50% trichloroacetate Denatured proteins were removed by centrifugation and the supernatants were neutralized with 74 lL of 2 m Tris⁄ HCl (pH 8.0) The concentration of 2-OG was determined in reaction mixtures containing 50 mm NaPO4 (pH 7.2), 0.2 mm NADH, 10 mm NH4Cl and 3 UÆmL)1 glutamate dehydrogenase from beef liver (Oriental Yeast, Tokyo, Japan) by measuring the absorbance change at 340 nm Pyruvate concentration was determined in a reaction buffer containing 1 UÆmL)1 lactate dehydrogenase from rabbit
Fig 4 Phylogenetic tree of AT3, T litoralis AGT homologues and subfamily Ic aminotransferases 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 trees were constructed using the neighbor-joining method and showed the same overall topology as the trees constructed by the maximum likelihood method In addition to the sequences in Fig 3, those from the following organ-isms were used: Desulfovibrio vulgaris (YP_010112), Halorhodospira halophila (YP_001001722), human (NP_872603), Hydrogenivirga sp 128-5-R1-1 (ZP_02176974), Hydrogenobaculum sp Y04AAS1 (YP_002121232), Nitrococcus mobilis (ZP_01127658), Pyrococcus horikoshii (1X0M_A), Sulfurihydrogenibium sp YO3AOP1 (YP_001931603) and Thermus thermophilus (BAC76939) AAAAT, a-aminoadipate aminotrans-ferase; KAT-II, kynurenine aminotransferase II.
Trang 8muscle (Roche, Basel, Switzerland) instead of NH4Cl and
glutamate dehydrogenase
For the kinetic assay of GOT activity in the direction of
Glu synthesis, a coupling method was applied using
thermostable malate dehydrogenase from Thermus flavus
(Sigma, St Louis, MO, USA) The reaction mixture
con-tained 50 mm NaPO4 (pH 7.2), 10 mm Asp, 5 mm
2-oxoglutarate, 0.2 mm NADH, 1 UÆmL)1malate
dehydro-genase and the enzyme solution The mixture was incubated
at 70C, and the absorbance was monitored at 340 nm to
estimate the decrease in NADH
Enzyme purification
AT1 was purified from 10 g of wet cells Active fractions
were selected according to GOT and GPT activities The
cells were washed with 20 mm Tris⁄ HCl buffer (pH 8.0) and
disrupted by sonication Cell debris was removed by
centri-fugation at 100 000 g for 1 h The supernatant, which was
designated CFE, was applied to a DE52 open column
(25 mm· 15 cm; Whatman, Brentford, Middlesex, UK)
equilibrated with 20 mm Tris⁄ HCl buffer (pH 8.0)
contain-ing 1 mm MgCl2 After the elution of bound proteins with
buffer containing 1 m NaCl, ammonium sulfate was added
to the fractions obtained to 30% saturation, and the samples
were applied to a Butyl-Toyopearl column (22 mm· 15 cm;
Tosoh, Tokyo, Japan) equilibrated with 20 mm Tris⁄ HCl
buffer (pH 8.0) containing 1 mm MgCl2and ammonium
sul-fate at 30% saturation This and subsequent
chromatogra-phy steps were performed using an A¨KTA purifier system
(GE Healthcare, Piscataway, NJ, USA) Proteins were eluted
with a gradient of ammonium sulfate from 30% to 0% over
230 mL at a flow rate of 4 mLÆmin)1 The active fractions
were dialysed against 20 mm Tris⁄ HCl buffer (pH 8.0)
con-taining 1 mm MgCl2, and were applied to a
DEAE-Toyo-pearl column (22 mm· 15 cm; Tosoh) equilibrated with
20 mm Tris⁄ HCl buffer (pH 8.0) containing 1 mm MgCl2
Proteins were eluted with a gradient of NaCl from 0 to 1 m
over 380 mL at a flow rate of 4 mLÆmin)1 The active
frac-tions were dialysed against 20 mm Tris⁄ HCl buffer (pH 8.0)
containing 1 mm MgCl2, and were applied to a MonoQ HR
5⁄ 5 column (bed volume, 1 mL; GE Healthcare) equilibrated
with 20 mm Tris⁄ HCl buffer (pH 8.0) containing 1 mm
MgCl2 Proteins were eluted with a gradient of NaCl from 0
to 1 m over 40 mL at a flow rate of 0.5 mLÆmin)1 The active
fractions were designated purified AT1, and stored at
)80 C until use
AT2 was purified from 20 g of wet cells Active fractions
were selected according to GGT and GPT activities CFE
was prepared from the cells and applied to the DE52 column,
Butyl-Toyopearl column and DEAE-Toyopearl column, as
described above The active fractions were applied to a CHT
Ceramic Hydroxyapatite column (16 mm· 11 cm; Bio-Rad,
Hercules, CA, USA) equilibrated with 1 mm KPO4 buffer
(pH 7.0) Proteins were eluted with a gradient of KPO4
buffer from 1 to 400 mm over 90 mL at a flow rate of 3 mLÆ min)1 The active fractions were dialysed against 20 mm Tris⁄ HCl buffer (pH 8.0) containing 1 mm MgCl2, and were applied to the MonoQ column in the same way as AT1 The active fractions were designated purified AT2, and stored at )80 C until use
AT3 was purified from 40 g of wet cells Active fractions were selected according to GGT activity CFE was pre-pared from the cells and applied to the DE52 column, Butyl-Toyopearl column, DEAE-Toyopearl column, CHT Ceramic Hydroxyapatite column and MonoQ column in the same way as AT2 Ammonium sulfate was added to the fractions obtained to 30% saturation, and the samples were applied to a Phenyl Superose column (bed volume, 1 mL;
GE Healthcare) equilibrated with 20 mm Tris⁄ HCl buffer (pH 8.0) containing 1 mm MgCl2and ammonium sulfate at 30% saturation Proteins were eluted with a gradient of ammonium sulfate from 30% to 0% over 15 mL at a flow rate of 0.5 mLÆmin)1 The active fractions were designated purified AT3, and stored at –80C until use
N-terminal amino acid sequencing The N-terminal amino acid sequences of purified amin-otransferases were determined by Procise 492HT (Applied Biosystems, Foster City, CA, USA) from a blotted mem-brane [0.2 lm Sequi-Blot poly(vinylidene) difluoride; Bio-Rad]
Protein assay Protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford, IL, USA) A calibration curve was plotted using bovine serum albumin as a standard protein
Gel filtration For the estimation of the molecular mass, gel filtration was performed using a Superose 6 HR 10⁄ 30 column (GE Healthcare) or a Shim-pack Diol-300 column (Shimadzu, Kyoto, Japan) equilibrated with 20 mm Tris⁄ HCl (pH 8.0) buffer containing 1 mm MgCl2 and 150 mm NaCl at flow rate of 0.5 or 1 mLÆmin)1, respectively Gel Filtration Stan-dard (Bio-Rad) was used as a molecular maker for calibra-tion Each measurement of standards or samples was performed in triplicate
Phylogenetic tree construction Amino acid sequences were aligned using the muscle program [33] After gap regions had been removed, phylo-genetic trees were constructed by the neighbor-joining method or the maximum likelihood method using phylip 3.67 [34]
Trang 9Nucleotide sequence accession numbers
Nucleotide sequences of AT1, AT2 and AT3 have been
deposited in the DDBJ⁄ EMBL ⁄ GenBank nucleotide
sequence database under accession numbers AB536750,
AB536751 and AB536752, respectively
Acknowledgement
This work was supported by a Grant-in-Aid for JSPS
Fellows (20-6284)
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Supporting information
The following supplementary material is available: Fig S1 Multiple sequence alignment of AT2, PSOT and AGT
Fig S2 Multiple sequence alignment of AT3, the
T litoralisAGT and family I aminotransferases This supplementary material can be found in the online version of this article
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