(Received for publication, September 13, 1995; and in revised form, October 13, 1995)
From the
The cDNA for the rat cytosolic branched chain aminotransferase
(BCAT) has been cloned. The BCAT
cDNA encodes a
polypeptide of 410 amino acids with a calculated molecular mass of 46.0
kDa. By Northern blot analysis, BCAT
message of
approximately 2.7 kilobases was readily detected in rat brain, but was
absent from liver, a rat hepatoma cell line, kidney, and skeletal
muscle. When expressed in COS-1 cells, the enzyme is immunologically
indistinguishable from the native enzyme found in rat brain cytosol.
Comparison of the rat BCAT
sequence with available data
bases identified the Escherichia coli (and Salmonella
typhimurium) branched chain aminotransferase (BCAT) and revealed a Haemophilus influenzae BCAT, a yeast BCAT, which is
hypothesized to be a mitochondrial form of the enzyme, and the murine
BCAT
(protein ECA39). Calculated molecular masses for the
complete proteins are 33.9 kDa, 37.9 kDa, 42.9 kDa, and 43.6 kDa,
respectively. The rat BCAT
sequence was 84% identical with
murine BCAT
, 45% identical with yeast, 33% identical with H. influenzae, 27% identical with the E. coli and S. typhimurium BCAT, and 22% identical with the evolutionary
related D-amino acid aminotransferase (D-AAT)
(Tanizawa, K., Asano, S., Masu, Y., Kuramitsu, S., Kagamiyama, H.,
Tanaka, H., and Soda, K.(1989) J. Biol. Chem. 264,
2450-2454). Amino acid sequence alignment of BCAT
with D-AAT suggests that the folding pattern of the
overlapping mammalian BCAT
sequence is similar to that of D-AAT and indicates that orientation of the pyridoxal
phosphate cofactor in the active site of the eukaryotic BCAT is the
same as in D-AAT. Thus, BCAT are the only eukaryotic
aminotransferases to abstract and replace the proton on the re face of the pyridoxal phosphate cofactor. Finally, requirements
for recognition of substrate L-amino acid and
-carboxylate binding are discussed.
In mammals, the branched chain aminotransferase (BCAT) ()catalyzes the first reaction in the catabolism of the
essential branched chain amino acids leucine, isoleucine, and valine.
Our laboratory has shown that there are two mammalian BCAT, a cytosolic
and mitochondrial isoenzyme, BCAT
and BCAT
,
respectively (Wallin et al., 1990). The rat BCAT
is found in brain, where it is the predominant isoenzyme, and in
placenta and ovary (Hutson, 1988; Hall et al., 1993; Ichihara,
1985). The second isoenzyme, BCAT
, is found in almost all
tissues of the body (Hutson et al., 1992). It has been shown
that BCAT
is a bifunctional protein catalyzing transport of
branched chain
-keto acids as well as transamination while
BCAT
exhibits only transaminase activity (Hutson and Hall,
1993).
Unlike mammals, bacteria appear to have a single BCAT. The
two mammalian BCAT isoenzymes are similar to the Escherichia coli and Salmonella typhimurium BCAT with respect to substrate
preference and kinetic properties (Inoue et al., 1988;
Lee-Peng et al., 1979; Hall et al., 1993); however,
the mammalian BCAT appear to be distinct from their ancestral
progenitor in both size and quaternary structure. The E. coli and S. typhimurium enzymes are hexamers composed of
identical 33.9-kDa subunits (Lipscomb et al., 1974). The
mammalian BCAT that have been purified are larger than 40 kDa, and
available data suggest that the functional unit of BCAT is
a homodimer while the functional unit of BCAT
may be a
monomer (Wallin et al., 1990; Hall et al., 1993;
Ichihara, 1985). No BCAT has been purified yet from lower eukaryotes
such as yeast.
Based on primary sequence comparisons with other aminotransferases, it was hypothesized that the prokaryotic BCAT and D-AAT are members of a unique evolutionary subclass of aminotransferases (Mehta et al. 1993; Tanizawa et al., 1989). Recently, Grishin et al.(1995) have placed these aminotransferases in a separate folding class (fold type IV) and identified 4-amino-4-deoxychorismate lyase and murine protein ECA39 as additional members of this group. Because of its role in providing D-amino acids for bacterial cell wall synthesis, D-AAT has been a target enzyme for development of novel antimicrobial agents (Sugio et al., 1995). The crystal structure of D-AAT, which has just been solved, has revealed that the folding pattern of this protein is completely different from those of other known PLP enzymes (Sugio et al., 1995). As discussed by Sugio et al. (1995), alignment of the bacterial BCAT and D-AAT amino acid sequences suggests that the folding pattern of the bacterial BCAT resembles that of D-AAT (Sugio et al., 1995). Understanding the relationship between the mammalian and the bacterial BCAT has been limited to date by the lack of cDNAs for the mammalian enzymes which has precluded structural analysis.
In this study, the cDNA for the rat cytosolic isoenzyme,
BCAT, has been isolated. The deduced amino acid sequence
revealed a 410-amino acid protein with a calculated molecular mass of
46,045 daltons. Sequence comparisons with available data bases
identified two additional full-length eukaryotic sequences including
the murine BCAT
(protein ECA39) and a yeast BCAT as well as
a bacterial BCAT from Haemophilus influenzae. The deduced
amino acid sequence for rat BCAT
shares considerable amino
acid sequence identity with the murine and yeast sequences and small,
but significant, identity with the bacterial BCAT and D-AAT.
Amino acid sequence alignment of BCAT
and D-AAT
firmly places the eukaryotic enzymes in fold type IV (Grishin et
al., 1995) and shows that the evolutionary conservation of key
residues involved in cofactor binding as well as provides clues to
amino acid residues that may be involved in substrate recognition.
Figure 5:
Multiple sequence alignment of eukaryotic
and prokaryotic branched chain aminotransferases (BCAT). The deduced
amino acid sequence of the rat BCAT was aligned with mouse
ECA39 (Mouse BCAT
), the S. cerevisiae twt1 gene (Yeast BCAT), H. influenzae Rd (H.
infl BCAT) and Ilve_E (E. coli BCAT) using the Multiple
Sequence Alignment software of the Genetics Computer Group Sequence
Analysis Software Package, version 8.0 (Devereux et al.,
1984). The BCAT accession numbers are: D21652 (murine), X78961 (yeast),
X02413 (E. coli), and HI1193 (H. influenzae Rd)
(Fleischmann et al., 1995). The active site lysine which forms
the internal aldimine with the pyridoxal phosphate cofactor is
indicated with a solid circle. The consensus sequence was
determined using a plurality of 5.0.
Rat brain cytosols from whole rat brain and isolated cerebellum, cortex, and hippocampus were prepared as described (Hall et al., 1993). Protein was determined in cytosol fractions and COS-1 cell extracts using the DC Protein Assay (Bio-Rad).
N-terminal amino acid sequence could not be obtained from
purified BCAT; therefore, two separate protein samples were
subjected to Lys-C endopeptidase and trypsin digestion, respectively.
The amino acid sequence of nine internal peptides is shown in Table 1. When the amino acid sequence of these peptides was
analyzed, a significant number of the peptides (Cyto 38, 54, 24, 44,
45, and 74; see Table 1) were found to align with a previously
identified mouse cDNA, ECA39, which was an overexpressed mRNA found in
the murine PCC4 Aza1 teratocarcinoma cell line (Niwa et al.,
1990). The rat BCAT
tryptic peptide sequence Cyto 74 and
two overlapping endopeptidase Lys-C peptides, Cyto 44 and Cyto 45,
showed 83% identity over a 29-amino acid region at the predicted
carboxyl terminus of the ECA39 protein. However, the rat BCAT
peptide sequence extended an additional 17 amino acids past the
end of the mouse-predicted coding sequence. Further analysis of the
ECA39 sequence revealed that upon shifting the reading frame by the
addition of a single nucleotide residue (guanosine) at position 1018,
the predicted coding sequence would now align with the three BCAT
peptide sequences. The amino acid identity was now greater than
90%, and all of the BCAT
peptides, except Cyto 48, could be
aligned with the mouse sequence. If a second nucleotide was inserted in
the codon at position 1262 to 1263, the deduced ECA39 sequence would
now align with Cyto 48 and place Cyto 48 at the C terminus of
BCAT
. This sequence alignment and expression of ECA39 in
brain and kidney (Niwa et al., 1990) strongly suggested that
ECA39 is the mouse homolog of BCAT
and explains why Grishin et al. (1995) placed it in the same folding class as the E. coli BCAT.
Using the nucleotide sequence of ECA39 and
peptide sequence derived from the purified rat BCAT,
nondegenerate oligonucleotide primers were designed and used in the
polymerase chain reaction with rat brain cDNA. A single product of the
expected size (630 base pairs) was obtained. Partial sequence analysis
of the product revealed at least 80% nucleotide identity with the mouse
candidate BCAT
. This product was used to screen a rat brain
gt10 cDNA library (approximately 1
10
plaques). Eight positives clones were identified. Six of the
original eight plaques were positive by polymerase chain reaction, and
two of these clones were plaque-purified and subcloned into
pBluescript.
The cDNA and deduced amino acid sequence for BCAT are shown in Fig. 1. The predicted initiator methionine
lies in an appropriate consensus for initiation of translation (Kozak,
1987) and is preceded by a 5`-untranslated region of 62 nucleotides.
The translation termination codon is followed by a 3`-untranslated
region of 72 nucleotides that did not extend to the poly(A) tail. The
rat cDNA encodes a protein of 410 amino acids with a calculated
molecular mass of 46,045 daltons which is in excellent agreement with
the molecular mass of 47 kDa determined by SDS-PAGE (Hall et
al., 1993). The translated BCAT
sequence contains an
alanine residue immediately following the N-terminal methionine. The
presence of a modified N-terminal alanine would be consistent with
reports characterizing the processing of amino termini of cytosolic
eukaryotic proteins (Flinta et al., 1986; Huang et
al., 1987) which have shown that when alanine is adjacent to the
N-terminal methionine, removal of the initiator methionine residue and
modification of the exposed alanine occurs with high frequency. The
location of the peptide sequences obtained from proteolytic digestion
of purified BCAT
in the translated sequence are indicated
in Table 1. A comparison of the amino acid sequences determined
chemically with those determined from the cDNA clone reveals 100%
agreement with the tryptic peptide sequences (Cyto 74 and 48) and Cyto
peptides 24, 41, and 54. The mismatches in Cyto 44 (His-283) and Cyto
45 (His-312) were correctly determined in Cyto 74 as glycine.
Chemically, glutamine was observed at position 344 in Cyto 27, and only
one serine instead of two was observed at position 70 in Cyto 38.
Figure 1:
Nucleotide and
predicted amino acid sequence of the rat BCAT. The
nucleotides and amino acids are numbered on the right of the
sequence. Amino acid residue 1 is the alanine residue immediately
following the predicted initiator methionine; an asterisk denotes the stop codon. The underlined nucleotide
residues indicate the location of primers used in the polymerase chain
reaction to generate a rat BCAT
probe.
BCAT is known to have a unique tissue distribution in
the rat. The enzyme is found in brain and is notably absent in other
tissues with the exception of low activity in ovary and placenta
(Hutson, 1988; Ichihara, 1985; Hall et al., 1993). To compare
BCAT
expression with the known tissue distribution of
BCAT
activity and protein, Northern blot analysis was
performed with total RNA from whole rat brain (see Fig. 2, lane B). Since the distribution of BCAT
throughout
the brain is not known, both BCAT
expression and cytosol
activity were examined in the cerebellum, cortex, and hippocampus (see Fig. 2, lanes marked C, H, and CE). A
single mRNA transcript of approximately 2.7 kb was found in all brain
regions. The additional length of the mRNA as compared to the length of
the cDNA most likely represents an additional 3`-untranslated region.
The putative murine BCAT
is also encoded by a larger mRNA
with a 1107-nucleotide 3`-untranslated region. BCAT
activity was found in all brain regions. The ubiquitous
expression of BCAT
in different brain regions is consistent
with reports showing branched chain transamination in astrocyte and
neuronal cultures (Hertz et al., 1987; Yudkoff et
al., 1994). What has not yet been determined is the exact
distribution of BCAT isoenzymes in different cell types in the brain.
Figure 2:
Northern blot hybridization of RNA from
rat brain. In the upper panel, total RNA (10 µg) from
whole brain (B), cerebellum (Ce), cortex (C), and hippocampus (H) was subjected to
electrophoresis, blotted, and hybridized using the uniformly P-labeled BCAT
cDNA probe (BCAT
) as described under
``Materials and Methods.'' The washed filter was exposed to
Fuji RX film with an intensifying screen for 2 days at -70
°C. The migration of RNA standards run in an adjacent lane are
indicated. Branched chain aminotransferase activity was measured in
cytosol fractions as described under ``Materials and
Methods.'' Aminotransferase activity is shown relative to the
activity in whole brain cytosol. Activity in whole brain cytosol was
51.7 ± 2.9 milliunits/mg of protein determined in four separate
cytosol preparations. In the lower panel, the filter was
stripped and rehybridized with a uniformly
P-labeled
glyceraldehyde-3-phosphate dehydrogenase probe (GAPD), washed,
and exposed to Fuji RX film for 2 days.
Fig. 3shows Northern blot analysis performed with total RNA
from a rat liver hepatoma (H4-II-E-C3) cell line, rat kidney, liver,
and skeletal muscle. Brain RNA is shown for reference. Consistent with
previous reports on the tissue distribution of rat BCAT (Hutson, 1988; Ichihara, 1985; Hall et al., 1993), no
hybridization was observed with RNA from these tissues, even after
lower stringency hybridization conditions. Stripping and reprobing the
blots with a probe for glyceraldehyde-3-phosphate dehydrogenase
(Ercolani et al., 1988) confirmed the load and integrity of
the RNA ( Fig. 2and Fig. 3, lower panels).
Figure 3:
Northern blot hybridization of RNA from
selected rat tissues. Ten micrograms of total RNA from whole brain (B), rat hepatoma cell line H4-II-E-C3 (He), liver (L), kidney (K), and mixed skeletal muscle (M) were subjected to electrophoresis and hybridized with the
uniformly P-labeled BCAT
cDNA probe (BCAT
) as described under
``Materials and Methods'' and in the legend to Fig. 2.
In the lower panel, the stripped filter was rehybridized with
a glyceraldehyde-3-phosphate dehydrogenase probe (GAPD).
To
demonstrate functional expression of BCAT, BCAT
cDNA was ligated into an expression vector (pBCAT
)
and transfected into COS-1 cells. The expressed BCAT
protein was visualized by immunoblotting using an antibody raised
against purified rat BCAT
(Hall et al., 1993) (see Fig. 4A). Cell extracts were also assayed for BCAT
activity and immunoprecipitated with BCAT
antiserum (see Fig. 4B). Lane 5 (pBCAT
) contains
the pBCAT
-transfected COS-1 cell extract, and lane 1 extract from mock-transfected COS-1 cells (p
Gal). Lanes
2-4 contain rat brain cytosol preparations (Brain
Cytosol) as a reference. A band of about 47 kDa was readily
detected in the rat brain cytosols and in the
pBCAT
-transfected COS-1 cell extract. The appearance of the
lower band in lane 5 varied according to the composition of
the inhibitor mixture which indicated that the lower band resulted from
proteolytic degradation. The same observation was made when the enzyme
was purified from rat brain cytosol which suggests that the protein is
very sensitive to proteolysis (Hall et al., 1993). COS-1 cell
BCAT specific activity was 26-fold higher (range 20- to 100-fold in
four separate transfection experiments) than in brain cytosol and
essentially all BCAT activity was neutralized by the BCAT
antiserum (see Fig. 4B, pBCAT
). Neither BCAT
antigen (see Fig. 4A, lane 1) nor significant BCAT activity
(see Fig. 4B, p
Gal) was observed in the
extract from the p
Gal-transfected cells. The relative amino acid
substrate specificity of BCAT
expressed in COS-1 cells with
KIC as amino acceptor was also examined. The amino acid preference in
descending order was isoleucine, leucine, valine, norvaline, glutamate,
and norleucine, which is essentially the same as has been reported
previously for purified BCAT
(Hall et al., 1993).
Phenylalanine and alanine were not transaminated (
1% of control
value with leucine as amino acceptor).
Figure 4:
Expression of rat BCAT in
pBCAT
-transfected COS-1 cells. A, cell extracts
(60 µg) from pCMV-
-galactosidase-transfected COS-1 cells (lane 1, p
Gal), pBCAT
-transfected
COS-1 cells (lane 5, pBCAT
), and
whole rat brain cytosol (100 µg) (lanes 2-4, Brain Cytosol) were subjected to SDS-PAGE and immunoblotted as
described under ``Materials and Methods.'' Protein molecular
mass standards are labeled Std. B, branched chain
aminotransferase activity was measured in cell extracts and brain
cytosol before (Control) and after immunoprecipitation with
BCAT
antiserum (Ab BCAT
). NM, not measured.
In addition to the murine
ECA39 cDNA, a search of the available protein and nucleic acid data
bases revealed that the rat BCAT protein sequence has
significant similarity to a yeast sequence, and partial sequences from
the nematode Caenorhabditis elegans, the plant Arabidopsis
thaliana, several human fragments as well as the bacterial BCAT
from E. coli and S. typhimurium and a gene product
from H. influenzae Rd (Fleischmann et al., 1995). A
multiple sequence alignment of the rat, mouse, yeast, and bacterial
BCAT sequences is shown in Fig. 5. Since only 7 of 308 residues
from the genetically similar E. coli (Ilv_E) and S.
typhimurium (Ilv_S) are different, and these differences do not
affect the consensus sequence, only the E. coli sequence was
used in the alignment. The deduced amino acid sequence from the H.
influenzae Rd BCAT, which is only 31% identical with the other two
bacterial sequences, was included as a separate entry in the alignment.
Rat BCAT
is significantly similar to the eukaryotic BCAT
exhibiting 84% and 45% identity to the murine and yeast sequences,
respectively. The rat sequence exhibits 27% identity with the E.
coli BCAT and 33% identity with the H. influenzae gene
product. Sequence identity with D-AAT from B. subtilis was 22%. On the other hand, comparison of the BCAT
sequence with the rat and porcine cytosolic aspartate
aminotransferases revealed only 18% and 16% sequence identity,
respectively.
Several important results have emerged from the
determination of the complete amino acid sequence of rat
BCAT. All the eukaryotic enzymes have molecular masses
greater than 40 kDa compared to just under 34 kDa for the E. coli and S. typhimurium enzymes. The calculated molecular mass
for the H. influenzae BCAT is intermediate at 37.9 kDa.
Without exception, the additional amino acid sequence is at the
N-terminal region of the proteins, and this is the most variable region
in these protein sequences (see Fig. 5). The consensus sequence
for the eukaryotic enzymes reflects the high degree of sequence
conservation with 208 residues in the consensus sequence (not shown).
This includes a sequence of 13 amino acids starting at BCAT
Leu-71 immediately preceding the N terminus of the E. coli BCAT in Fig. 5. When the bacterial BCAT are included, 57
amino acids remain in the consensus sequence. The PLP binding site of
rat brain BCAT
is most likely Lys-246 with the conserved
sequence KXGXNY found in all BCAT sequences including
the mitochondrial isoenzyme. (
)Other regions showing
relatively high sequence identity in all BCAT enzymes include the
sequence of 14 amino acids beginning at BCAT
Leu-112 and
the sequence of 12 amino acids beginning at Gly-356. BCAT
Phe-135, Trp-92, and Trp-405 are also conserved in all BCAT
sequences.
This manuscript presents the first cloning of a mammalian
BCAT. The tissue distribution of the BCAT mRNA is
consistent with the known distribution of the cytosolic BCAT isoenzyme
determined by measurements of activity and immunological techniques.
The protein, when expressed in COS-1 cells, is immunologically
indistinguishable from the enzyme in rat brain cytosol, and the amino
acid substrate preferences were the same as the enzyme purified from
rat brain cytosol (Hall et al., 1993).
Two additional
complete eukaryotic BCAT sequences have been identified. The ECA39 cDNA
(Niwa et al., 1990), once two nucleotide changes are made,
appears to code for the murine BCAT with a calculated
molecular mass of 42.9 kDa. The other sequence is a yeast BCAT with a
calculated molecular mass of 43.6 kDa for the complete protein. Very
little is known about the yeast BCAT including whether or not these
lower eukaryotes contain both isoenzymes. However, the calculated
isoelectric point for the yeast enzyme of 9.59 is considerably higher
than the calculated values of 5.6 and 5.25 for the rat and mouse
cytosolic enzymes, respectively. Hartmann and Christen(1991) observed
that, with few exceptions, the pI of the mitochondrial isoenzyme has a
more basic isoelectric point than the cytosolic form of the same
enzyme. If the yeast enzyme is a mitochondrial protein, then it is
likely to have an N-terminal targeting sequence. The alignment of the
mammalian BCAT with the yeast sequence becomes unambiguous at
BCAT
-51 (Yeast BCAT-25) and Leu-71 in
BCAT
(Yeast BCAT-45) is the start of a highly
conserved sequence (LVFG in Fig. 5). Mitochondrial
targeting signals are generally from 10-70 amino acids, can form
an amphipathic helix composed of positively charged and hydrophobic
residues and generally lack acidic amino acids (Hartl et al.,
1989). An arginine is usually found at the -2 position from the
mitochondrial processing peptidase cleavage site (Hartl et
al., 1989). Secondary sequence analysis shows potential helical
structure in this region, and an
helix would be amphipathic. Only
one acidic residue, an aspartic acid, is found in the first 34 amino
acids. Based on the alignment shown in Fig. 5and assuming an
arginine at the -2 position, cleavage could occur in the yeast
BCAT between residues 16 and 17 resulting in a mature protein of 377
amino acids with a calculated molecular mass of 41 kDa. Two other
potential cleavage sites preceding BCAT
Leu-71 appear less
likely since they occur after BCAT
-51.
Cloning of the
first mammalian BCAT, rat BCAT, and identification of two
additional full-length eukaryotic sequences, is the first step in
developing a molecular model for the eukaryotic BCAT. Sequence
similarity between the E. coli BCAT and D-AAT led to
the hypothesis that these two enzymes evolved from a common ancestral
gene (Tanizawa et al., 1989). The enzymes are 25% shorter than
the average aminotransferase length of approximately 400 amino acids,
and alignment of these enzymes with other aminotransferases indicates
that the small domain as defined in aspartate aminotransferase is
either absent or of different structure. Another unique property of
these enzymes concerns the stereochemistry of the hydrogen transfer
which occurs during catalysis on the re face of the cofactor
while in other classes of aminotransferases proton transfer occurs from
the si face (Yoshimura et al., 1993). The crystal
structure of the pyridoxamine-form of thermostable D-AAT has
been solved, and it is clear that the structure is distinct from the
other PLP-containing enzyme structures that have been reported (Sugio et al., 1995).
Fig. 6presents a GAP alignment of
rat BCAT and D-AAT. Structural analysis of D-AAT shows that, unlike other aminotransferases, the
N-terminal domain (D-AAT residues 1-120) and the
C-terminal domain (D-AAT residues 121-282) are comprised
of very different secondary and tertiary structures, and the pyridoxal
phosphate binding site is located mainly at the interface of the two
domains of the same monomer rather than being shared between the two
monomers as in aspartate aminotransferases (Sugio et al.,
1995). Only two residues, Arg-98* and His-100* from the other subunit,
extend to the active site and PLP-binding region. Secondary structure
predictions of the overlapping regions of rat BCAT
and D-AAT show a similar pattern, suggesting that the two proteins
have similar folding patterns. Therefore, the mammalian BCAT can be
classified as belonging in the folding group IV described by Grishin et al. (1995).
Figure 6:
Alignment of rat cytosolic branched chain
aminotransferase (BCAT) with D-amino acid aminotransferase (D-AAT). The deduced
amino acid sequences of the rat BCAT
and thermostable BacillusD-AAT were aligned using the GAP analysis
software (Genetics Computer Group Sequence Analysis Software Package,
Version 8.0) (Devereux et al., 1984). A Bestfit analysis did
not give a significantly different alignment. A single manual
adjustment was made at D-AAT-215 to preserve the
-helix
in D-AAT (Sugio et al., 1995). Structurally important
residues in D-AAT, conserved residues in folding class IV
proteins, and residues that are also conserved in the BCAT
sequence are boxed. The asterisk denotes that
Arg-98* and His-100* are from the opposite subunit in the D-AAT dimer. Residues discussed in relation to
-carboxylate binding are indicated by arrows.
Since the components necessary for
transamination are clearly present in the shortest bacterial BCAT, the
function of the N-terminal region of the eukaryotic enzymes is not
known. Except for the conserved sequence around BCAT-71,
the N-terminal portions of the eukaryotic BCAT are the most variable
regions of these sequences (see Fig. 5). Secondary structure
predictions for the N terminus of BCAT
are predominantly
-helix, suggesting an ordered structure. Since the quaternary
structure of rat BCAT
and the E. coli BCAT appears
to be different, dimer versus hexamer, the possibility exists
that the N-terminal extension may influence quaternary structure. (The H. influenzae Rd BCAT appears to be of intermediate size, but
the protein's quaternary structure is not known.) Another
possibility is that the small highly conserved portion of this region
(BCAT
-71 to -82) serves a function analogous to the
extended amino segment of the aspartate aminotransferases and
stabilizes the dimer (Fukumoto et al., 1991). The N terminus
may also play a role in domain movement (Kirsch et al., 1984).
In the transamination reaction the catalytic lysine has an important
role in determining the stereochemistry of the proton transfer (Kirsch et al., 1984; Sugio et al., 1995). In L-aspartate aminotransferase, the lysine faces the side of the
substrate where the -proton is located (see Fig. 7A). The lysine is the general base involved in
abstraction of the proton from the
-carbon and reprotonation of
the aldehydic carbon of the coenzyme to yield a ketimine intermediate.
The lysine then returns the proton onto the pro-S side of the
substrate so that the L-configuration is maintained when the
reaction proceeds in the reverse direction. The lysine approaches the
coenzyme from the si face which is the side facing the
protein. In D-AAT and E. coli BCAT, the proton is
added to or abstracted from the C4` atom of the coenzyme-imine or
external aldimine intermediate on the re face (Yoshimura et al., 1993). The crystal structure of D-AAT shows
that the cofactor is bound so that the side chain of the catalytic
lysine (D-AAT Lys-145) and the
-proton point toward the
protein as in L-aspartate aminotransferase (see Fig. 7B). Key active site residues involved in cofactor
binding in D-AAT are conserved in the bacterial BCAT
suggesting a similar cofactor orientation (Fig. 7C).
The GAP alignment shown in Fig. 6indicates that these
structural features are conserved in rat BCAT
and also in
the other BCAT (see Fig. 5). The key residues in D-AAT
that are also found in BCAT
include the active site lysine (D-AAT Lys-145), D-AAT Glu-177, which interacts with
the pyridoxal nitrogen, and the residues which interact with the
phosphate group, D-AAT Ile-204, Thr-205, Thr-241, and Arg-50
(Sugio et al., 1995) (see boxed residues in Fig. 6). Since it is the main chain amide of D-AAT
Ile-204 that forms the hydrogen bond with a phosphate oxygen,
substitution of a Val (BCAT
-313) in this position should
not perturb the structure significantly. The helix dipole (N terminus
of helix D-AAT 203-216) which interacts with the
negative charge on the phosphate may also be conserved in the BCAT (see Fig. 6). Secondary structure predictions for the equivalent
residues in BCAT
, residues 312-325, suggest that
BCAT
and D-AAT would have similar secondary
structure in this region. D-AAT Leu-201, which is about 3.9
Å from the C4` of PLP, is also conserved in BCAT
(Leu-310 in Fig. 6) and the other BCAT sequences (see Fig. 5). The D-AAT L201A and L201W mutants show
anomalous kinetics, and the enzymes appear to form an inactive
pyridoxamine form during catalysis (Kishimoto et al., 1995).
In addition, two other residues conserved in fold class IV enzymes, D-AAT Gln-181 and Phe-183, are also found in BCAT
.
Therefore, it seems reasonable to conclude that orientation of the PLP
cofactor in the eukaryotic BCAT is the same as in D-AAT (see Fig. 7, B and C).
Figure 7:
Schematic diagram of the orientation of
the substrate amino acid, active site lysine, and pyridoxal phosphate
(PLP) cofactor in L-aspartate aminotransferase (ASP-AT), D-amino acid aminotransferase (D-AAT), and L-branched chain aminotransferases (BCAT). The cofactor is positioned as seen in the structure of
porcine Asp-AT, D-AAT, and the predicted position in the rat
BCAT. The cofactor side facing solvent is toward the viewer
and the si face facing the protein (ASP-AT), and, in D-AAT and BCAT, the re face is facing the protein.
The incoming
-amino acid reacts with the internal aldimine between
the active site lysine and PLP, and the
-proton faces the
protein.
Two elements of the PLP
binding site in D-AAT may be altered in the BCAT enzymes.
Tyr-31 in D-AAT which appears to be in a position to bind to
the phenolic oxygen (O3`) and imine nitrogen atom of PLP is replaced by
phenylalanine in all BCAT (see Fig. 5and Fig. 6). The
equivalent tyrosine in L-aspartate aminotransferase plays an
important role in stabilization of intermediates during transamination
(Kirsch et al., 1984; Arnone et al., 1985). Whether
or not the tyrosine in the adjacent conserved motif beginning at
BCAT Leu-112 (LHYxxx(L,v,c)FEG) would be in a
position to hydrogen-bond the phenolic oxygen remains to be
established. A second difference in D-AAT and aspartate
aminotransferase is in the residues that form a parallel stacking
interaction with the pyridoxal ring. The tryptophan residue that serves
this function in aspartate aminotransferase is absent in D-AAT. Instead, the side chain hydroxyls of a loop of three
serine residues (D-AAT Ser-179 to Ser-181) hydrogen-bonded to
the carboxylate group of D-AAT Glu-166 form a barrier on one
side of the pyridoxal ring. None of these residues appears to be
conserved in any of the BCAT (see Fig. 5and Fig. 6).
Although several tryptophan residues are conserved in the eukaryotic
BCAT sequences, only BCAT
Trp-92 and Trp-405 are conserved
in all BCAT. These two tryptophans would presumably be found in the
BCAT small and large domains, respectively. Thus, it would appear that
the BCAT have evolved a different structural solution in this region of
the protein.
D-AAT accepts a broad range of D-amino acids. On the other hand, the mammalian BCAT are
selective for L-branched chain amino acids and their straight
chain analogs, but exhibit no activity with alanine or aromatic amino
acids (Hall et al., 1993; Ichihara, 1985). As shown in Fig. 7, B and C, the orientation of the
-hydrogen dictates that the
-carboxyl and side chain of an L-amino acid in the BCAT active site must be in the opposite
orientation from a D-amino acid in the substrate binding
pocket. Two elements of the substrate binding site include a
``carboxylate trap'' which is positively charged and a
``side-chain pocket'' to discriminate substrate species
(Kirsch et al., 1984; Arnone et al., 1985). In D-AAT, the substrate
-carboxylate is thought to interact
with Arg-98* (see Fig. 6) of the other subunit dimer. No
arginine is present in the BCAT consensus sequence around the D-AAT Arg-98. This difference is predictable, because of the
opposite orientation of an L-amino acid and D-amino
acid in the active site (see Fig. 7, B and C);
hence, this region of the BCAT is probably involved in side chain
recognition. Modeling of E. coli BCAT on the D-AAT
structure that is described, but not shown in Sugio et
al.(1995), led to the proposal that either the Arg present in the E. coli sequence equivalent to residue D-AAT-35 or D-AAT-88 could be located on the proper side of the active
site for
-carboxylate binding in the bacterial enzyme. Examination
of the alignment of BCAT
with D-AAT in Fig. 6and the consensus sequence in Fig. 5does not
reveal a conserved arginine residue at either position in the BCAT
sequences although four arginines are found in the consensus sequence
shown in Fig. 5. However, there is a conserved arginine at
BCAT
-187 (equivalent to D-AAT-83) and an arginine
that is found in all BCAT sequences except the E. coli enzyme
at BCAT
-126 (D-AAT-38) (see Fig. 6).
Mutagenesis will be required to test these candidate arginines.
In summary, cloning of the eukaryotic BCAT has confirmed placement of the BCAT in the same folding group as D-AAT and the bacterial BCAT (Grishin et al., 1995). The mammalian proteins, however, have acquired additional amino acid sequence during evolution, a structural change that has not been observed in the other aminotransferase subclasses (Mehta et al., 1993). Preliminary modeling of the mammalian BCAT on the D-AAT structure (data not shown) has provided some insights into regions that may be important in the structure of the BCAT active site which can be tested by site-directed mutagenesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35774[GenBank].