(Received for publication, August 7, 1995; and in revised form, October 6, 1995)
From the
We report here the cloning and sequencing of the cDNA,
purification, steady state kinetic analysis, and truncation mapping
studies of the human 5-aminoimidazole-4-carboxamide ribonucleotide
formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase). These enzyme
activities catalyze the penultimate and the final steps of de novo purine biosynthesis, respectively. In all species of both
prokaryotes and eukaryotes studied, these two activities are present on
a single bifunctional polypeptide encoded on the purH gene.
The human purH cDNA is 1776 base pairs in length encoding for
a 591-amino acid polypeptide (M = 64,425).
The human and avian purH cDNAs are 75 and 81% similar on the
nucleotide and amino acid sequence level, respectively. The K
values for AICAR and
(6R,6S)10-formyltetrahydrofolate are 16.8 µM ± 1.5 and 60.2 µM ± 5.0, respectively,
for the cloned, purified human enzyme. A 10-amino acid sequence within
the COOH-terminal portion of human AICARFT/IMPCHase has some degree of
homology to a previously noted ``folate binding site.''
Site-directed mutagenesis studies indicate that this sequence plays no
role in enzymatic activity. We have constructed truncation mutants
which demonstrate that each of the two enzyme activities can be
expressed independent of the other. IMPCHase and AICARFT activities are
located within the NH
-terminal 223 and COOH-terminal 406
amino acids, respectively. The truncation mutant possessing AICARFT
activity displays steady state kinetic parameters identical to those of
the holoenzyme.
Aminoimidazole ribonucleotide formyltransferase (AICARFT) ()catalyzes the penultimate step of the de novo purine biosynthetic pathway (Fig. 1). Along with another
enzymatic activity earlier in the pathway, glycinamide ribonucleotide
formyltransferase (GARFT), it requires a reduced folate cofactor,
10-formyltetrahydrofolate. Interest in AICARFT stems in part from its
potential as a chemotherapeutic target. Inhibitors of GARFT are
currently in clinical trials as anti-neoplastic agents. Also, AICARFT
inhibition is thought to be the origin of the anti-purine effects of
anti-folates such as methotrexate whose primary target is dihydrofolate
reductase.
Figure 1: AICARFT and IMP cyclohydrolase reactions of de novo purine biosynthesis.
In all organisms studied to date, AICARFT activity is accompanied by inosine monophosphate cyclohydrolase (IMPCHase, also known as inosinicase) located on the same polypeptide encoded by the purH gene. IMPCHase is the final step in the purine de novo pathway. The activities of the purine pathway in eukaryotes are frequently found on multifunctional proteins, whereas in bacteria, the enzymes are typically monofunctional proteins. Thus, AICARFT/IMPCHase is an exception in being bifunctional throughout evolution. A question which arises is whether there is some particular advantage which favors the bifunctional arrangement. For example, there might be kinetic ``channeling'' of the intermediate, formamidoimidazole ribonucleotide (FAICAR), between the penultimate and final catalytic centers. It is also possible that the enzyme could have a single binding site for the nearly complete purine ribonucleotide, which would be sequentially operated upon by two catalytic sites. Alternatively, there may be two functionally independent domains, similar to the situation with the other multifunctional proteins of the de novo purine pathway as it exists in eukaryotes.
AICARFT/IMPCHase from Bacillus subtilis(1) , Salmonella typhimurium(2) , Escherichia
coli(3) , and from chicken (4) have been cloned
and expressed. Since there are no monofunctional proteins for sequence
comparison, nothing is known about potential domain structure for
AICARFT/IMPCHase from any species. Although GARFT and AICARFT/IMPCHase
utilize the same cofactor and carry out very similar reactions, there
has been no recognized sequence homology between them. This report
describes the cloning, sequence, expression, and purification to
homogeneity of the human AICARFT/IMPCHase. Comparison of the human
sequence with those of other species revealed a high degree of sequence
conservation and homology. In addition, an amino acid sequence can be
located which corresponds to a ``folate binding consensus
sequence'' (5) previously noted in other
10-formyltetrahydrofolate binding enzymes such as GARFT. However, in
contrast to other such sequences, this does not help to locate the
catalytically active amino acid residues in AICARFT/IMPCHase and
appears to be simply a common structural element. Truncation mutant
studies identify a COOH-terminal fragment which possesses no IMPCHase
activity but does possess AICARFT activity kinetically equivalent to
the holoenzyme. An NH-terminal fragment has also been
identified which has IMPCHase activity but lacks AICARFT activity.
These results suggest that AICARFT and IMPCHase activities exist as
independent domains.
Figure 2: Human AICARFT/IMPCHase cDNA clone. PCR was used to assemble the full-length 1776-bp human purH cDNA. Three overlapping partial human purH cDNA clones were obtained as presented under ``Experimental Procedures.'' A, 5` purH cDNAs pHAT84 and pHAT4A were joined by PCR resulting in a 705-bp human 5` purH cDNA fragment. B, this 705-bp cDNA fragment was used in a subsequent PCR reaction with the 3` 1142-bp cDNA fragment. Unique NcoI and BamHI restriction sites (in brackets) were introduced via oligonucleotide design. A conservative amino acid replacement, S2A, was introduced for cloning purposes.
Cell lysates were
clarified by centrifugation at 4 °C, 39,000 g for
40 min in a Beckman Ti70 rotor. The clarified lysate was subsequently
applied to a reactive red 120-agarose (Sigma) column (2.5
12.0
cm) equilibrated in Buffer HB. The column was washed with 10 column
volumes Buffer HB. Human AICARFT/IMPCHase was eluted using 20 mM Tris-Cl, pH 7.5, 500 mM NaCl, 50 mM KCl, 20%
glycerol. Fractions were assayed for either AICAR formyltransferase or
IMP cyclohydrolase activity. Peak activity fractions were pooled and
were diluted with an equal volume of ice-cold Buffer HB. The enzyme was
subsequently concentrated using an Amicon ultrafiltration cell (250 ml)
with a Diaflo YM30 ultrafiltration membrane (Amicon, Inc.). A buffer
exchange into Buffer HB was then performed, and the enzyme was
concentrated to a final volume of 10-15 ml. In order to further
purify human AICARFT/IMPCHase, the enzyme was desalted using a G25
coarse Sepharose (Pharmacia Biotech Inc.) column (1.5
22.0 cm)
equilibrated in Buffer B (20 mM Tris-Cl, pH 7.5, 20%
glycerol). AICARFT/IMPCHase was immediately applied to an
AICAR-Sepharose column (1.5
5.5 cm) equilibrated in Buffer B.
AICAR-Sepharose was prepared according to the method of Smith et
al.(9) using commercially available CNBr-activated
Sepharose 4B (Pharmacia). The enzyme was eluted with 10 mM AICAR, 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 50
mM KCl, 20% glycerol. Fractions possessing IMPCHase activity
were pooled and concentrated using Centricon 30 concentrators. A buffer
exchange was performed into Buffer B. Typically, the enzyme was passed
over the AICAR-Sepharose column a second time in order to further
purify the AICARFT/IMPCHase. This second AICAR-Sepharose column elution
was also followed by Centricon 30 enzyme concentration with a buffer
exchange into Buffer HB. In order to determine if all AICAR had been
removed, AICARFT activity assays were performed in the presence and
absence of additional AICAR.
Purified protein and protein fractions
were resolved throughout the purification by discontinuous
SDS-polyacrylamide gel electrophoresis as described by
Laemmli(10) . Typical running and stacking gels used were 12%
(w/v) and 4% (w/v), respectively. An SDS reducing sample buffer (62.5
mM Tris-Cl, pH 6.8, 10% glycerol, 2% SDS, 0.71 M -mercaptoethanol, 0.0013 (w/v) bromphenol blue) was added to
protein samples prior to electrophoresis. Samples were subsequently
heated to 90 °C for 5 min, loaded onto the SDS-polyacrylamide gel,
and electrophoresed. Gels were stained with Coomassie Blue R250.
Molecular
mass standard curves for the Superdex 75 column were calibrated using
the elution profiles of the standard molecular mass markers
(Pharmacia): albumin (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsin
(25.0 kDa), and ribonuclease A (13.7 kDa) monitored by ultraviolet
absorbance at = 280 nM. The column void volume
was determined by the elution of dextran blue (2,000 kDa) from the
column.
Reaction mixtures
(500 µl = final reaction volume) contained a final
concentration of 33 mM Tris-Cl, pH 7.4, 25 mM KCl, 5
mM 2-mercaptoethanol, 0.1 mM (6R,6S)-10-formyltetrahydrofolate and 0.05
mM AICAR as described by Mueller and Benkovic(15) .
The reaction mixture was mixed prior to the addition of the human
AICARFT/IMPCHase enzyme and subsequently mixed again. AICARFT activity
was monitored by the formation of FH at A
for 4 min according to the method of Black et al.(16) employing the use of a Perkin-Elmer Lambda2
spectrophotometer. The concentration of FH
formed was
calculated by the difference in values between the extinction
coefficients for 10-formyl-FH
and FH
, 19.7
10
M
cm
(16) .
The first
NH-terminal AICARFT/IMPCHase cDNA truncation mutant,
pETHAT-IMP, was constructed by PCR amplification of the first 669
nucleotides using the upstream and downstream primers
5`-CGG(CCATGG)CTTCTCTATCAGCCTTATTTAG and
5`-GAGAT(GCGGCCGC)GGGCTGCAGTGTGTACAGC, respectively (unique NcoI and NotI restriction endonuclease sites are in
parentheses and the start codon underlined. A conservative amino acid
change S2A, indicated in bold, was introduced to facilitate cloning and
expression of human AICARFT/IMPCHase). The PCR product was resolved on
a 1.2% agarose gel, gel-purified, and cloned into pCR(TM)II
(Invitrogen). The resulting plasmid, pTAIMP, was digested with NcoI and NotI, and the 669-bp 3` purH
fragment was subsequently directionally subcloned into the NcoI and NotI polylinker sites of expression vector
pET-23d creating plasmid pETHAT-IMP.
A second truncation mutant expressing the amino-terminal 230 amino acid residues was constructed. However, rather than cloning into the His-tag(TM) vector, pET-23d, we used vector pET-14b. Unique NcoI and BamHI restriction sites (designated in parentheses) were introduced into the 5` and 3` ends via oligonucleotide design. Truncation mutant pETHUIMP-230 was constructed using the upstream primer 5`-CGG(CCATGG)CTTCTCTATCAGCCTTATTTAG (described previously) and the downstream primer 5`-CCCAA(GGATCC)TCATAGAACTGTGATGGGAAGC. Again, start and stop (antisense) codons are underlined.
The carboxyl-terminal truncation mutant, pETHATNB-1200, was generated using a similar strategy. The upstream and downstream primer combinations used include: 5`-GGA(CCATGG)CAATTTCAGATTATTTC/5`-CCCAT(GGATCC)TCAGTGGTGGAAGAGCCGAAGGTTC. Unique NcoI and BamHI restriction sites, indicated in parenthesis, were introduced into the 5` and 3` ends via oligonucleotide design. Start and stop (antisense orientation) codons are underlined. This recombinant was similarly constructed in vector pET-14b.
Figure 3: Nucleotide sequence of human AICARFT/IMPCHase cDNA and derived amino acid sequence of the protein. The nucleotide sequence is numbered from the initiator codon in the cDNA.
Figure 4: Comparison of the amino acid sequences of eukaryotic and prokaryotic AICARFT/IMPCHase proteins. The aligned sequences are human (HU), avian (AV), E. coli (EC), S. typhimurium (ST), and B. subtilus (BS). The alignment was made using the PILEUP program (Genetic Computer Group, Madison, WI). Darkly shaded regions represent amino acid residues that are strictly conserved. Lighter shaded areas represent those residues having a single base change on the nucleic acid level and are, therefore, noted as representing ``mutationally'' conservative amino acid changes.
Figure 5: SDS-polyacrylamide gel of human AICARFT/IMPCHase fractions during purification. Enzyme purification of the human AICARFT/IMPCHAse, 64.4 kDa, expressed from the cDNA is described under ``Experimental Procedures.'' Enzyme fractions indicated are: total cellular protein from E. coli expressing human AICARFT/IMPCHase from recombinant pETHATNB-1800 (lane 1), total soluble protein from E. coli extracts expressing human AICARFT/IMPCHase from recombinant pETHATNB-1800 (lane 2), pooled AICARFT/IMPCHase from the reactive red column step (lane 3), AICARFT/IMPCHase following Amicon concentration of reactive red enzyme pool (lane 4), G25 desalting of the human enzyme (lane 5), first AICAR-Sepharose purification (lane 6), second AICAR-sepharose purification step (lane 7). Molecular mass standards include: phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; carbonic anhydrase, 31.0 kDa; and soybean trypsin inhibitor, 21.5 kDa.
The steady state
kinetic parameters for the purified human AICARFT/IMPCHase were
determined (Table 2). The AICARFT activity followed
Michaelis-Menton kinetics for the determination of the K values for both AICAR as well as 10-formyltetrahydrofolate. These
kinetic data are consistent with values published for the native enzyme
purified from MCF-7 human breast cancer cells (20) as well as
the values obtained from human CCRF-CEM cells from two independent
sources (
)(21) .
We also conducted experiments to
determine the K for FAICAR. Consistent with
results reported by Mueller and Benkovic (15) for the avian
enzyme, our data for the human AICARFT/IMPCHase indicate that the K
for FAICAR is <1 µM. The
spectrophotometric assay for inosinicase activity is not sensitive
enough to accurately measure values below 1 µM.
Figure 6: Truncation mutants of human AICARFT/IMPCHase independently expressing the two activity domains. The full-length cDNA(pETHATNB-1800), and each of the three truncation mutants (pETHUIMP-230, pETHAT-IMP, pETHATNB-1200) are depicted. The portions of the human purH coding region expressed in pET vectors are indicated by lines. The sizes of the DNA and the corresponding expressed polypeptides are indicated in parentheses. Both AICAR formyltransferase and IMP cyclohydrolase enzyme activity assays were performed on extracts prepared from E. coli transformed each of the recombinants as described under ``Experimental Procedures.'' Enzyme activity (+) and no enzyme activity(-) are indicated.
Figure 7:
Proposed 10-formyl-FH binding
site. Depicted is an alignment of the human AICARFT/IMPCHase, human
AICARFT/IMPCHase (HU AICARFT) (amino acids 469-503), and
the human GARFT (HU GARFT) (amino acids 915-951). The
10-formyl-FH
-binding site hypothesized by Cook et al.(5) is indicated in brackets (HU GARFT amino
acids H915-G924). GARFT active site residues, His
and
Asp
are indicated by asterisks. Corresponding
amino acids in the human AICARFT/IMPCHase chosen for mutagenic studies
include His
, Asp
, and
Asp
.
Amino acid sequence alignment of the human GARFT domain with the human AICARFT/IMPCHase indicate that this sequence is at least partially conserved. It was necessary to introduce a number of gaps in order to maximize the similarities between the human GARFT and AICARFT/IMPCHase amino acid sequences in the region under investigation. In addition, there are potential secondary structural differences between the human GARFT and AICARFT/IMPCHase sequences. GARFT possesses a proline residue which is not conserved within the human AICARFT/IMPCHase sequence. Moreover, this putative consensus sequence is conserved among the two known eukaryotic PurH sequences but not among any of the known prokaryotic PurH sequences. Nevertheless, these similarities warranted investigation.
Amino acid residues His and
Asp
, essential residues for GARFT activity, correspond to
human AICARFT/IMPCHase residues His
and
Asp
. It was noted that AICARFT/IMPCHase Asp
was also conserved and due to its proximity to Asp
we also chose to study that residue as well. We investigated
whether these residues were essential for either human AICARFT or
IMPCHase activities using site directed mutagenesis. Three separate
point mutations resulting in conservative amino acid replacements
H469A, D501N, and D503N were each introduced into the human purH cDNA. Each of these three mutants, pETHAT-H469A,
pETHAT-D501N, and pETHAT-D503N, as well as the wild type cDNA clone
pETHATFLNN-1 and the vector pET-23d were expressed in E. coli.
Protein expression was analyzed by SDS-polyacrylamide gel
electrophoresis. Extracts prepared from each of the three mutants as
well as the wild type purH cDNA clone indicated that a
64.3-kDa polypeptide was overexpressed corresponding to the size of
full-length human PurH (data not shown). Both AICARFT and IMPCHase
activity assays were performed using each of the extracts. AICARFT as
well as IMPCHase activity was detected in extracts prepared from
transformants possessing the wild type as well as each of the three
mutant clones. No AICARFT or IMPCHase was detected in extracts prepared
from the pET-23d transformant. These data indicate that conservative
amino acid residue changes of amino acid residues His
,
Asp
, and Asp
do not inactivate either
AICARFT or IMPCHase activity. The enzyme activity of mutant
pETHAT-H469A diminished more rapidly with storage than the wild type or
either of the other two mutants. While mutation H469A does not
eliminate either of the two enzyme activities of human PurH, it may
create structural perturbations that result in enzyme instability.
The sequence of the human purH cDNA bears marked
similarity to the previously reported avian sequence (4) with
75 and 81% identity on the nucleotide and amino acid sequence levels,
respectively. The human sequence also demonstrates similarity to purH from prokaryotes, with amino acid identities ranging from
31 to 36% among the three bacterial sequences
reported(1, 2, 3) . While these sequences are
similar, it should be noted that there appear to be distinct
differences that may be characterized as being either more
eukaryotic-like (human PurH amino acids
Ile-Leu
and residues
Tyr
-Lys
) or more prokaryotic-like
(Gap intrajected between human PurH Phe
and
Arg
) (Fig. 4).
The steady state kinetic values for the AICARFT reaction using the human protein expressed in E. coli are comparable with those reported for native PurH derived from several human cell types. Thus, postsynthetic modification of the protein does not appear to be important for activity. The physical as well as the kinetic properties of the cloned human enzyme are likely to be similar to that of the native enzyme.
Enzyme cross-linking experiments for the avian enzyme conducted by Mueller and Benkovic (15) suggest that the avian AICARFT/IMPCHase may form a dimer. Under the conditions employed thus far, evidence indicates that the human enzyme exists as a monomer. It is of course possible that the human enzyme might form a dimeric or higher aggregate species under other conditions. We are continuing to investigate this possibility.
A question of major interest is whether the AICARFT and IMPCHase
activities exist as separate functional domains or whether the two
activities might share a common substrate binding site. To address
this, we constructed a number of separate truncation mutants. Among
these were found two NH-terminal mutants, 223 and 230 amino
acids in length, each of which lacked AICARFT activity but showed
IMPCHase activity. IMPCHase activity can thus be localized within the
first 223 amino acid residues of human AICARFT/IMPCHase. An additional
truncation mutant expressing the COOH-terminal 406 amino acid residues
possessed solely AICARFT activity. The degrees of overlap between each
of the two NH
-terminal mutants and COOH-terminal truncation
mutant are 37 and 44 amino acids. Construction of smaller amino- or
carboxyl-terminal truncation mutants resulted in loss of IMPCHase and
AICARFT activity, respectively. Although it is possible that the small
overlap region might include a common substrate binding site, this
seems unlikely in view of its small size. It seems more likely that the
bifunctional protein contains two separate functional domains. These
might or might not be connected by a linking sequence. The next
smallest NH
-terminal truncation mutant we have tested is
164 amino acids in length and shows no IMPCHase activity. The next
smallest COOH-terminal truncation mutant is 364 amino acids in length
and is likewise devoid of activity. Since the possibilities of unstable
or misfolded proteins can not be ruled out, these mutants may not
completely define the limits of the putative functional domains.
A further intriguing question is whether or not the two domains are functionally independent or whether the intermediate, FAICAR (Fig. 1), might be ``channeled'' to the IMPCHase domain. The COOH-terminal truncation mutant demonstrated independent function with kinetic properties highly similar to those of the holoenzyme. The available spectrophotometric assay for IMPCHase activity lacks the sensitivity necessary to permit comparison of activities between the truncation mutants and the holoenzyme. Thus, the question of potential channeling must await the development of a more sensitive assay.
We investigated a region of the human
AICARFT/IMPCHase sequence corresponding to the putative
10-formyl-FH binding domain of human GARFT and other
10-formyl-FH
-requiring enzymes using site-directed
mutagenesis. Conservative amino acid replacements were introduced into
the human purH cDNA by site-directed mutagenesis. These amino
acid replacements had no effect on either AICARFT or IMPCHase enzyme
activities for any of the mutant enzymes. While the residues
His
and Asp
are essential for GARFT
activity, the corresponding residues in the human AICARFT/IMPCHase are
not essential. Therefore, this region may represent a common structural
motif among these 10-formyl-FH
-requiring enzymes but is not
involved in the single carbon transfer reaction nor in the binding of
the folate substrate for AICARFT/IMPCHase. The crystal structure of E. coli GARFT shows that this region is near the folate
binding site and may be important in the enzyme
mechanism(25, 26, 27) . Since no additional
regions of similarity of the primary amino acid sequence between these
two 10-formyl-FH
-requiring enzymes are apparent, the actual
site of 10-formyl-FH
binding for AICARFT/IMPCHase remains
to be elucidated. Further investigation utilizing active site
irreversible inhibitors against either AICARFT activity or inosinicase
activity will aid in the identification of residues essential for these
activities.
Chemical modification studies by Szabados et
al.(21) suggest that the IMPCHase activity of the human
PurH requires an essential cysteine as well as an essential arginine
residue. Amino acid sequence alignment of all known PurH sequences
indicates that there are no cysteine residues conserved among all of
the sequences (Fig. 4). There are, however, a total of 9
cysteine residues in the human AICARFT/IMPCHase, each of which is
conserved in the avian AICARFT/IMPCHase sequence as well. Two of these
cysteine residues, Cys and Cys
, are
localized within the amino-terminal truncation mutant expressing human
IMPCHase activity. Based upon our truncation mutant studies, three
arginine residues serve as potential candidates essential for IMPCHase
activity. These arginine residues, Arg
, Arg
,
and Arg
(numbered relative to the human sequence), are
conserved among all known PurH sequences and are localized in large
conserved ``blocks'' within the putative IMPCHase activity
domain (Fig. 4). Residue Arg
is located within the
37 amino acid overlap between our two truncation mutants possessing
either IMPCHase activity or AICARFT activity and may be a candidate for
mutational studies. Other potential candidates include Arg
and Arg
which are conserved in all PurH sequences
with the exception of Bacillus subtilis where there is a
proximal arginine residue
Expression of the cloned human purH cDNA in E. coli has permitted production of large quantities of human AICARFT/IMPCHase. Further investigations of the structure and mechanism of this enzyme are now feasible.