Kynurenine aminotransferase (EC 2.6.1.7, KAT(
))
catalyzes the irreversible transamination of the L-tryptophan
metabolite L-kynurenine to form kynurenic acid. Due to an
overlapping substrate specificity, multiple forms of pyridoxal
5`-phosphate (PLP)-dependent aminotransferases are apparently able to
catalyze this reaction(1) . Differences probably exist in the
various tissues and among different animal species regarding which
enzyme form is predominantly responsible for the biosynthesis of
kynurenic acid. A soluble form of KAT has recently been identified and
cloned from rat(2, 3) , and it was found that the
amino acid sequence of this pyruvate-preferring form of KAT is
identical to that reported for rat kidney cysteine S-conjugate
-lyase(4) , also referred to as glutamine transaminase K
(GTK, EC 2.6.1.64)(5) . The human form of this aminotransferase
with KAT, GTK, and
-lyase activity has also been cloned and shown
to have 82% amino acid similarity to the rat protein(6) .
In
rat kidney, a 2-oxoglutarate-preferring aminotransferase with KAT
activity has also been described. This protein corresponds to
-aminoadipate aminotransferase (AadAT, EC 2.6.1.39), an enzyme
involved in the metabolism of lysine (1, 7, 8, 9) that catalyzes the
reversible transamination reaction between L-2-aminoadipate
and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate.
Due to the postulated role of kynurenic acid as a putative
endogenous modulator of glutamatergic neurotransmission (for review,
see (10) ), particular attention has recently been devoted to
the presence of KAT isoenzymes in cerebral tissues. Kynurenic acid is,
in fact, an antagonist at the glycine site of N-methyl-D-aspartate receptors, and increased levels
of kynurenic acid may exert a neuroprotective action in some
pathological conditions(11) . Two different aminotransferases
able to produce kynurenic acid appear to be present in human brain (12, 13, 14) . A cDNA clone from rat brain
encoding an aminotransferase with KAT activity has been recently
isolated(3) , and it has been found that this protein
corresponds to the rat kidney
-lyase/GTK enzyme cloned by Perry et al.(4) . Rat brain also contains a mitochondrial
form of this aminotransferase, which differs from cytosolic KAT/GTK
exclusively for the presence of an NH
-terminal leader
peptide, which targets the protein to the mitochondrial
matrix(15) . Whereas it has been claimed that in rats, a single
pyruvate-preferring enzyme with KAT activity may be of physiological
relevance in the cerebral synthesis of kynurenic
acid(16, 17) , whether in rat brain other
aminotransferase forms are involved in the biosynthesis of kynurenic
acid remains to be established. Noteworthy, the presence of AadAT
activity has also been described in rat brain (see (18) and
references therein), therefore suggesting that this aminotransferase
may represent a second synthetic enzyme for kynurenic acid in the
central nervous system of this animal species.
In the present work,
we describe the molecular cloning and the functional expression of a
soluble aminotransferase from rat kidney displaying both KAT and AadAT
activity (KAT/AadAT).
MATERIALS AND METHODS
Enzymatic Activity Determination
KAT
activity was assayed as described previously(3) . For routine
analysis, aliquots of the enzyme preparation were incubated (1 h at 37
°C) in the presence of 1 mML-kynurenine and 1
mM 2-oxoglutarate in a final volume of 200 µl of 150
mM Tris acetate buffer, pH 8.0, containing 70 µM PLP. Kynurenic acid formed was quantified by HPLC with
spectrophotometric detection at 330 nm(3) . 3-Hydroxykynurenine
aminotransferase activity was determined using L-3-hydroxykynurenine as substrate and by measuring the
production of xanthurenic acid by HPLC with UV detection at 340 nm. AadAT activity was assayed as described in (19) . Briefly,
after incubation of the enzyme preparation in the presence of 1.7
mM 2-oxoadipate and 16.7 mML-glutamate,
2-oxoglutarate formed was measured using the glutamate dehydrogenase
assay. Aminotransferase activity toward other L-amino acids
was measured essentially as described previously(14) .
Kinetic constants were calculated by fitting the experimental data
to Michaelis-Menten equation using a computer program (Ultrafit,
Biosoft).
Purification of KAT/AadAT from Rat
Kidney
Rat kidneys were homogenized in 10 mM potassium phosphate buffer, pH 7.4 (containing 150 mM NaCl, 10 mM 2-mercaptoethanol, 50 µM PLP,
0.5 mM EGTA, 0.5 mM phenylmethanesulfonyl fluoride, 1
µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin),
with an Ultra-Turrax homogenizer. After centrifugation at 45,000
g, the supernatant was subjected to heat treatment (1
min at 60 °C) and to ammonium sulfate fractionation. The protein
was then purified by chromatography on Fast-Flow DEAE-Sepharose 6B
(Pharmacia Biotech Inc.), followed by hydrophobic interaction
chromatography on phenyl-Sepharose and by ion exchange on a Mono Q HR
5/5 FPLC column (Pharmacia). KAT/AadAT was eventually submitted to
chromatofocusing on a Mono P FPLC column (Pharmacia) (see legend to Fig. 1).
Figure 1:
Separation of rat
kidney KAT/AadAT and KAT/GTK by (A) DEAE-Sepharose
chromatography and (B) chromatofocusing. A, after the
ammonium sulfate precipitation step, the enzyme preparation in 10
mM potassium phosphate buffer, pH 7.4, containing 0.5 mM EGTA and 2 µM PLP, was applied onto a Fast-Flow
DEAE-Sepharose column. Elution was performed at 500 µl/min with a
linear gradient of potassium phosphate buffer, pH 7.4 (up to 250
mM). B, KAT/AadAT and KAT/GTK (Mono Q step), in 25
mM bis-Tris, pH 7.2, were injected onto a Mono P column
equilibrated in the same buffer. The column was eluted (700 µl/min)
with Polybuffer 74 (Pharmacia) adjusted to pH 5.0 with HCl. The
generated pH gradient is shown. Aliquots of the chromatographic
fractions were assayed for KAT activity with 1 mML-kynurenine using either 1 mM 2-oxoglutarate
(KAT/AadAT,
) or 1 mM pyruvate (KAT/GTK,
).
Protein content was measured by the Pierce Coomassie
Plus protein assay kit.
Rat Kidney KAT/AadAT Digestion, Peptide Mapping, and
Amino Acid Sequencing
The purified protein (
100
µg) was subjected to SDS-polyacrylamide gel-electrophoresis on a
12.5% mini-gel and subsequently electroblotted onto a polyvinylidene
difluoride membrane (Immobilon-P
, Millipore) (20) . After Ponceau S staining, digestion of blotted KAT/AadAT
with modified trypsin (Promega, Zürich,
Switzerland) and extraction of the resulting peptides were performed as
described previously(20, 21) . KAT/AadAT tryptic
peptides were then separated by reverse-phase HPLC (for details on the
chromatographic conditions used, see (3) ) and directly
subjected to amino acid sequence analysis by means of an Applied
Biosystem model 475A protein sequenator (Forster City, CA) with on-line
phenylthiohydantoin-derivative detection.
Preparation of Poly(A)
RNA
Total RNA was extracted from different adult rat
tissues by the guanidinium isothiocyanate/cesium chloride
method(22) . Poly(A)
RNA was obtained by two
purification cycles on oligo(dT)-cellulose spun columns (Pharmacia).
Isolation of Rat Kidney KAT/AadAT cDNA by
RT-PCR
RT reactions were carried out in 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl
, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 20 units of RNase inhibitor, 100 pmol of
antisense primer, 1 µg of rat kidney poly(A)
RNA,
and 50 units of cloned Moloney murine leukemia virus reverse
transcriptase (Perkin Elmer Corp.), in a final volume of 20 µl at
42 °C for 60 min. PCR was performed in a final volume of 100 µl
containing 20 µl of the reverse transcription reaction, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM
MgCl
, 0.2 mM dATP, 0.2 mM dCTP, 0.2
mM dGTP, 0.2 mM dTTP, 100 pmol of sense primer, and
2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). Cycling was
performed at 95 °C (1 min), 56 °C (1 min), and 72 °C (1
min) for 35 cycles. Degenerate oligonucleotide primers were synthesized
by Genosys Biotechnologies Inc. (The Woodlands, TX).The final PCR
product was electrophoresed on a 1% agarose gel. The major band of 1058
base pairs (bp) was cut from the gel, subcloned into the SmaI
site of the vector pBC/CMV(23) , and sequenced.
Screening of cDNA Library
A rat kidney
cDNA library (Uni-ZAP
XR) was purchased from Stratagene
and screened following previously described procedures (24) .
Colonies were grown overnight on duplicate nitrocellulose filters (HA
0.45 µm, Millipore). After prehybridization for 4 h at 42 °C,
the filters were hybridized with a
P-labeled
nick-translated 1058-bp rat kidney cDNA probe (5
10
cpm/ml) for 16 h in 6
NET solution (900 mM NaCl,
90 mM Tris-HCl, 6 mM EDTA, pH 8.3), 10
Denhardt's solution (0.2% bovine serum albumin, 0.2% Ficoll, 0.2%
polyvinylpyrrolidone), 20% deionized formamide, 1 mM sodium
pyrophosphate, 0.1% SDS, 100 mM ATP, 7 µg/ml
poly(A)
RNA, 6 µg/ml Escherichia coli DNA, and 500 µg/ml denatured salmon sperm DNA. Following
washing for 30 min at room temperature in 6
SSC (900 mM NaCl, 90 mM sodium citrate, pH 7.0) and a 1-h wash in 6
SSC containing 0.5% SDS, the filters were dried and exposed to
Kodak X-Omat
AR film with intensifying screens.From a
total of
9
10
plaques, 10 positive clones were
obtained and purified through an additional round of screening at lower
plaque density. Bluescript plasmids (pBSK, Stratagene), carrying the
cDNA inserts, were then isolated from positive phages via in vivo excision.
DNA Sequencing
All DNA sequences were
determined by the method of Sanger et al.(25) , as
modified for plasmid double-stranded DNA sequencing (Sequenase version
2.0; U. S. Biochemical Corp.). Sequencing from the plasmid vectors, T3
and T7 Bluescript primers, and a set of consecutive inner primers were
used. DNA sequence assembly, analysis, and translation were performed
using the Gene Jockey 1.3 software package (Biosoft Ltd., Cambridge,
United Kingdom).
Northern Blot Analysis
Poly(A)
RNAs (3 µg) isolated from various rat tissues were
electrophoresed on a 1.1% agarose gel containing formaldehyde. After
vacuum blotting (PosiBlot, Stratagene) onto an Amersham
Hybond-N
filter (Amersham Life Science, Little
Chalfont, UK, RPN 1520N), RNAs were then cross-linked by UV
irradiation. After prehybridization for 3 h at 42 °C, the RNA blot
was hybridized for 20 h with a
P-labeled nick-translated
rat kidney cDNA probe (10
cpm/ml) in 50% deionized
formamide, 5
SSC (750 mM NaCl, 75 mM sodium
citrate, pH 7.0), 0.1% SDS, 10% dextran sulfate, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, and 50 µg/ml denatured salmon sperm DNA. The
filters were washed once with 2
SSC (300 mM NaCl, 30
mM sodium citrate, pH 7.0) for 30 min at room temperature and
finally with 1
SSC (150 mM NaCl, 15 mM sodium
citrate, pH 7.0) containing 0.5% SDS for 1 h at 55 °C.
Expression of Recombinant Rat Kidney
KAT/AadAT in HEK-293 Cells
The 1.8-kilobase cDNA
insert encoding rat kidney KAT/AadAT was subcloned into the blunt-ended SmaI site of the expression vector pBC/CMV(23) ,
placing transcription of the cDNA under control of the strong immediate
early promoter of human cytomegalovirus. Human embryonic kidney
fibroblast cells (HEK-293 cell line, ATCC CRL 1573) were transfected
with sense and antisense cDNA as described previously(20) . 2
days after transfection, the cells were harvested, washed twice with
phosphate-buffered saline, and stored at -80 °C until
analysis. For activity determination of the recombinant enzyme, the
transfected cells were resuspended in 0.5 ml of 15 mM Tris
acetate buffer, pH 8.0 (containing 10 mM 2-mercaptoethanol,
0.5 mM phenylmethanesulfonyl fluoride, 50 µM PLP), and homogenized in a Polytron homogenizer (Kinematica AG).
After centrifugation at 28,000
g for 20 min at 4
°C, aliquots of the supernatant were assayed for enzymatic activity
as described above.
RESULTS AND DISCUSSION
Purification of Rat Kidney KAT/AadAT and
Internal Peptide Sequencing
KAT/AadAT was purified from rat
kidney following the procedure described in Table 1. The
purification of the enzyme was monitored by measuring KAT activity in
the presence of 2-oxoglutarate, its preferred aminoacceptor. The
purified enzyme also displayed AadAT activity with a specific activity
of 9.4 µmol min
mg protein
.
After chromatography on DEAE-Sepharose, this aminotransferase form was
well separated from KAT/GTK, a distinct enzyme with KAT activity and
preference for pyruvate as cosubstrate (Fig. 1). In accordance
with previous reports on rat kidney KAT
isoforms(9, 26) , FPLC chromatofocusing indicated pI
values of approximately 6.4 and 5.4 for KAT/AadAT and KAT/GTK,
respectively (see also (3) ). The native molecular mass of
KAT/AadAT was in the 90-100-kDa range (as determined by gel
filtration on a Pharmacia Superose 12 FPLC column) and was composed of
two subunits of equal size. In fact, SDS-polyacrylamide gel
electrophoresis analysis of the purified protein under reducing
conditions showed a single major band of
45 kDa (not shown).
To
obtain information on the amino acid sequence of KAT/AadAT, the
polyvinylidene difluoride-blotted protein was directly submitted to
Edman degradation to see if its NH
terminus was accessible
to sequencing. No sequence information could be obtained, indicating
that the NH
terminus of the protein was modified either as
the result of a posttranslational event or due to protein handling. The
amino acid sequences of internal peptides were then obtained after
digestion with trypsin and separation of the resulting peptides by
reverse-phase HPLC. The sequence of six tryptic peptides (denominated
T2a, T2b, T3, T6, T10, and T12, see Fig. 2and Fig. 3)
was determined. No significant matches were found in the Swiss-Prot and
Protein Identification Resource Protein data banks.
Figure 2:
Isolation of rat kidney KAT/AadAT cDNA
fragment by RT-PCR. A, the amino acid sequences of two tryptic
peptides from purified rat kidney KAT/AadAT are shown. The regions of
peptides that were selected for the synthesis of degenerate
oligonucleotides are overlined. B, degenerate sense (S) and antisense (A) oligonucleotide primers
corresponding to the peptide sequences in A are indicated
(Y
C/T, W
A/T, S
G/C, R
A/G; I indicates inosine). C, two combinations of sense and antisense oligonucleotides (lane 1, sense-T10/antisense-T3; lane 2,
sense-T3/antisense-T10) were used in RT-PCRs of rat kidney
poly(A)
RNA. RT-PCR products from each reaction (lanes 1 and 2) were resolved by agarose gel
electrophoresis. The arrow points to a 1058-bp fragment
containing the KAT/AadAT protein sequence.
Figure 3:
Nucleotide and predicted amino acid
sequence of cloned rat kidney KAT/AadAT. The deduced amino acid
sequence of the encoded polypeptide is shown in single-letter code below the nucleotide sequence in bold face type and
is numbered beginning with the initiating methionine. Nucleotides are
numbered in the 5` to 3` direction. The tryptic peptides (T6, T3, T2a,
T2b, T12, T10) isolated from rat kidney KAT/AadAT are boxed.
The asterisk denotes the 3`-terminal stop codon. Nucleotide
variation between several clones is indicated above the
appropriate nucleotide (C
T at nucleotide position 145 (rkKAT-14);
C
T at nucleotide position 1784
(rkKAT-8)).
Isolation of Rat Kidney KAT/AadAT
cDNA
Using the amino acid sequences of the analyzed tryptic
peptides of purified rat kidney KAT/AadAT, we were able to obtain the
corresponding cDNA using RT-PCR. Two amino acid sequences from the
least degenerate regions of peptides T3 and T10 were selected to
construct synthetic degenerate oligonucleotides. Because the relative
order of the two tryptic peptides within the protein was not known,
degenerate sense and antisense oligonucleotides corresponding to the
tryptic peptide sequences were synthesized (Fig. 2, panels A and B). Poly(A)
RNA extracted from rat
kidney was reverse-transcribed using each degenerate antisense
oligonucleotide as a primer, and the resulting cDNA was used as a PCR
template with each possible combination of degenerate sense and
antisense oligonucleotides from the two tryptic peptides. After 35
rounds of amplification, one distinct PCR product was detected by
agarose gel electrophoresis for the primer combination
sense-T3/antisense-T10 (Fig. 2C, lane 2). DNA
sequence analysis revealed that the 1058-bp fragment contained an open
reading frame coding for a polypeptide of 352 amino acids, starting
with the codon of the first amino acid phenylalanine (TTT) of peptide
T3 and ending with the second nucleotide of the last glutamine codon
(CA) of the T10 sequence. Moreover, this cDNA fragment encoded the
tryptic peptides T2a, T2b, and T12, confirming that it was indeed a
partial cDNA for rat KAT/AadAT.
Molecular Cloning of Rat Kidney KAT/AadAT
cDNA
A rat kidney cDNA library (Uni-ZAP
XR)
from Stratagene was screened with the partial cDNA probe of rat kidney
KAT/AadAT. Screening of 9
10
recombinants revealed
10 positive clones with inserts ranging from 950 to 1828 bp. The four
longest clones, designated as rkKAT-8, rkKAT-14, rkKAT-3, and rkKAT-9,
were chosen for further characterization. DNA sequence analysis showed
that all clones were identical, except for two nucleotide differences.
Only one of these nucleotide differences was contained within the
coding region, and it did not result in a change in amino acid
sequence. The nucleotide and deduced amino acid sequences are shown in Fig. 3. Analysis of the combined DNA sequence (nucleotides
1-1828) indicated the presence of a single 1275-bp open reading
frame with a predicted initiation codon (ATG) at nucleotide 113 and
termination at nucleotide 1388 with the stop codon TAA. This open
reading frame codes for a polypeptide of 425 amino acid residues with a
predicted molecular mass of 47,789 Da. It is preceded by a
112-nucleotide 5`-untranslated region containing two in-frame stop
codons at positions 32 and 71, and a 422-nucleotide untranslated region
in the 3`-end. The predicted initiation codon (nucleotide 113) is
embedded in the sequence GAGACATG, which does not match
perfectly with the consensus sequence CCACCATG frequently
found for eukaryotic translation initiation(27) . No further
ATG was found upstream of this predicted initiation codon, indicating a
complete coding sequence.Comparison of the complete sequence with
the EMBL DNA sequence data base using the Genetics Computer Group
sequence analysis software (GCG, University of Wisconsin), indicated
that the sequence was unique and had not been isolated previously.
Analysis of the hydrophilicity plot of the predicted amino acid
sequence for KAT/AadAT showed no evidence for membrane-anchoring or
-spanning regions, consistent with the soluble nature of the isolated
protein. A mitochondrial form of KAT/AadAT has also been
described(8, 9) . However, no structural features
resembling those of leader peptides for mitochondrial import (28) were observed in the predicted amino acid sequence of rat
KAT/AadAT. Therefore, whether mitochondrial KAT/AadAT is encoded by a
different gene or by a splice variant of the isolated form carrying an
additional signal-peptide sequence remains to be established. Four
potential N-linked glycosylation sites (Asn, Xaa, Ser/Thr) (29) were found in the predicted amino acid sequence of rat
kidney KAT/AadAT at Asn residues 2, 57, 101, and 202. No information,
however, is presently available indicating whether the native protein
is glycosylated.
KAT/AadAT Sequence Comparison with Other
Aminotransferases
Binary alignment of the protein sequences
of KAT/AadAT and KAT/GTK (4) using the GAP program contained in
GCG showed only 18.4% amino acid identity with 14 gaps located mainly
in the NH
- and COOH-terminal parts of the two sequences.
Considering conservative amino acid substitutions, the two KAT isoforms
displayed 46.2% similarity (not shown). Similar degrees of amino acid
identity and similarity were observed after comparison with other
aminotransferases from different species, including isoenzymes, such as
human serine:pyruvate aminotransferase(30) , which have been
reported to display KAT activity. Sequence homology among
aminotransferase isoenzymes is not easily recognizable by standard
algorithms for sequence comparison. A multiple sequence alignment of
aspartate aminotransferases from various organisms with KAT/AadAT and
KAT/GTK showed that several residues in the central region of the
sequences are totally conserved, therefore suggesting that the
corresponding three-dimensional structures might also be conserved. In
fact, despite the low degree of amino acid identity, most
aminotransferases appear to constitute a group of structurally
homologous proteins that originated from a single universal ancestor
protein(31) . Fig. 4shows an alignment of KAT/AadAT and
KAT/GTK with E. coli aspartate aminotransferase (AAT), whose
three-dimensional structure has been solved in detail. The structure of E. coli AAT consists of three parts, an
NH
-terminal loose segment and two compact domains, and
shows the same fold and active site structure of vertebrate isoenzymes
( (32) and references therein). The alignment of the larger
domain of E. coli AAT (from Thr
to
Ser
, Swiss-Prot data base numbering) shows only 15.5 and
13.3% sequence identity with KAT/AadAT and KAT/GTK, respectively,
whereas the sequence identity between the two KAT isoforms in this
region is 21.2%. A preliminary model for this domain of KAT/AadAT and
KAT/GTK was built by exchanging the amino acids of E. coli AAT
for those of the two KAT isoenzymes according to the sequence alignment
shown in Fig. 4and optimized using the Moloc molecular modeling
software (Moloc)(33) . For both isoenzymes, the scores
calculated by the three-dimensional profile method (34) indicated that the model exhibits features of a correctly
folded protein. According to the model obtained from the sequence
alignment, Lys
of KAT/AadAT and Lys
of
KAT/GTK correspond to the lysine residue (position 246) of the E.
coli enzyme, which forms an aldimine linkage with the PLP aldehyde
group. In addition, several of the AAT residues involved in the binding
of the cofactor (32) appear to be conserved in KAT/AadAT and
KAT/GTK (see Fig. 4). Sequence similarities in the
NH
- and COOH-terminal regions were too low for molecular
modeling. This suggests that KAT/AadAT and KAT/GTK should resemble AAT
in the large domain comprising the active site but may be different in
the domain composed by the NH
and COOH termini and possibly
involved in the substrate specificity.
Figure 4:
Sequence alignment of KAT/AadAT and
KAT/GTK from rat with E. coli AAT. Asterisks indicate
residues conserved among the three sequences. Amino acid identities in
KAT/AadAT and KAT/GTK are overlined. The residues involved in
cofactor binding (32) are shaded. The lysine residue
forming the aldimine bond with PLP is indicated in bold face
type. Note that the Swiss-Prot data base numbering is used for E. coli AAT.
Blot Hybridization
Northern blot analysis
of poly(A)
RNAs isolated from various rat tissues is
shown in Fig. 5. In all tissues tested, a single species of
KAT/AadAT mRNA of approximately 2.1 kilobases was detected using a
P-labeled nick-translated cDNA from rkKAT-8, indicating
that the in vivo mature KAT/AadAT mRNA may be longer than our
cloned cDNA, possibly in the 5`-untranslated region. Whereas labeling
intensity was highest for mRNA isolated from kidney, a clearly
detectable band of equal size was also observed in poly(A)
RNA extracted from total brain and cerebral cortex, indicating
the presence of this enzyme form in cerebral tissues. The weaker
labeling intensity found in brain in comparison to peripheral tissues
is in accordance with the much lower activity of KAT isoenzymes
detected in cerebral tissues (see e.g.(13) ). The
fact that no KAT/AadAT message was detected in the hippocampus might be
due to the low mRNA level for this protein in this brain region.
Figure 5:
Northern blot hybridization of
poly(A)
RNAs from various rat tissues. The blot was
hybridized with a
P-labeled nick-translated cDNA insert
isolated from rkKAT-8. Numbers on the left indicate kilobases (kb) as determined by RNA size markers (Life Technologies,
Inc.). The blot was exposed to x-ray film for 5 days (kidney and liver)
or 15 days (brain tissues).
Expression of Recombinant Rat KAT/AadAT in
HEK-293 Cells and Characterization of the Enzymatic
Activity
To confirm that the isolated cDNA indeed encoded
for an aminotransferase with both KAT and AadAT activity, the rat
KAT/AadAT cDNA was subcloned into the eukaryotic expression vector
pBC/CMV (23) . HEK-293 cells were chosen for transfection
because they did not exhibit either KAT or AadAT activity. A relatively
high expression of both KAT and AadAT activities could be reached after
transient transfection of these cells, with most of the enzymatic
activity (>90%) being recovered in the soluble fraction of the
cells. No activity was observed after transfection of the cells with
the antisense cDNA. Determination of KAT kinetic properties of the
recombinant enzyme with 2 mM 2-oxoglutarate as amino acceptor
showed K
and V
values for L-kynurenine of 0.95 ± 0.33 mM and 135
± 31 nmol min
mg protein
,
respectively. The enzyme also metabolized L-3-hydroxykynurenine to xanthurenic acid with a similar
catalytic efficiency (K
, 1.36 ± 0.16
mM; V
, 166 ± 42 nmol
min
mg protein
). In the presence
of 5 mML-kynurenine as amino donor, the K
of 2-oxoglutarate was 45.5 ± 12.4
µM. When the enzyme was assayed for AadAT activity, the K
for L-glutamate was 5.6 ± 2.8
mM, with a V
of 910 ± 210 nmol
min
mg protein
. Notably, under
our experimental conditions, the catalytic efficiency of KAT/AadAT
appeared to be similar for both KAT and AadAT activity, as it can be
inferred from the similar V
/K
ratios. The observed kinetic parameters are in accordance with
those found for the rat native
enzyme(1, 8, 19) .The KAT activity of the
enzyme measured in the presence of various cosubstrates was highest
with 2-oxoglutarate and 2-oxoadipate and lowest with pyruvate (Table 2). Regarding the specificity of this aminotransferase
form toward other L-amino acids, tryptophan, phenylalanine,
tyrosine, aspartate and alanine were found to be substrates for
KAT/AadAT. However, the specific activities observed in the presence of
these amino acids (up to 10 mM) were relatively low, being 15%
(tryptophan), 9% (phenylalanine and tyrosine), and 6% (aspartate and
alanine) of the activity measured with L-kynurenine. The
enzyme did not display significant enzymatic activity toward the other L-amino acids tested (histidine, serine, methionine,
glutamine, and leucine). The specificity pattern toward amino acids and
cosubstrates of the KAT/AadAT expressed in HEK-293 cells was similar to
that observed for the native enzyme purified from rat kidney.
The
availability of the cDNA clone for this aminotransferase isoenzyme with
KAT activity will contribute to the further clarification of kynurenic
acid disposition in peripheral organs as well as in the central nervous
system. For instance, in rat brain it appears that at least two
distinct aminotransferases with KAT activity are expressed: 1)
KAT/AadAT, as described in this paper, and 2) the KAT/GTK form, which
was directly isolated from cerebral tissues(3, 15) .
Several factors may determine the role of the various KAT forms in
kynurenic acid biosynthesis, such as the different affinity constants
of these enzymes for L-kynurenine and the regional and
cellular distribution of these enzymes in the different organs.
Interestingly, several biochemical characteristics of the two KAT forms
identified in rat, including pI value and cosubstrate preference, are
similar to those described for the two cerebral KAT forms in
humans(12, 13) , therefore raising the possibility
that these human KAT forms may be similar to the identified KAT forms
from rat.
Although our attention has focused mainly on the role of
KAT/AadAT in L-tryptophan metabolism, its function in L-lysine metabolism also merits further investigation, for
instance, in disorders of the lysine metabolic pathway. Interestingly, L-2-aminoadipate, the substrate for AadAT, is a well known
astroglial-specific toxin (18) . Thus, knowledge of the
cerebral disposition of this compound is instrumental for the
elucidation of its mechanism of toxicity and possible relevance in
pathology.