(Received for publication, June 27, 1994; and in revised form, November 28, 1994)
From the
We have identified a gene (iadA) in Escherichia
coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic
cleavage of L-isoaspartyl, or L--aspartyl,
dipeptides. We demonstrate at least a 3000-fold purification of the
enzyme to homogeneity from crude cytosol. From the amino-terminal amino
acid sequence obtained from this preparation, we designed an
oligonucleotide that allowed us to map the gene to the 98-min region of
the chromosome and to clone and obtain the DNA sequence of the gene.
Examination of the deduced amino acid sequence revealed no similarities
to other peptidases or proteases, while a marked similarity was found
with several dihydroorotases and imidases, reflecting the similarity in
the structures of the substrates for these enzymes. Using an E.
coli strain containing a plasmid overexpressing this gene, we were
able to purify sufficient amounts of the dipeptidase to characterize
its substrate specificity. We also examined the phenotype of two E.
coli strains where this isoaspartyl dipeptidase gene was deleted.
We inserted a chloramphenicol cassette into the disrupted coding region
of iadA in both a parent strain (MC1000) and a derivative
strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of
isomerized proteins. We found that the iadA deletion does not
result in reduced stationary phase or heat shock survival. Analysis of
isoaspartyl dipeptidase activity in the deletion strain revealed a
second activity of lower native molecular weight that accounts for
approximately 31% of the total activity in the parent strain MC1000.
The presence of this second activity may account for the absence of an
observable phenotype in the iadA mutant cells.
L-Aspartyl and L-asparaginyl residues are two
of the most prominent sites for the spontaneous decomposition of
proteins. These residues can undergo nonenzymatic intramolecular
reactions resulting in the formation of deamidated, racemized, and
isomerized derivatives (Clarke et al., 1992; Stephenson and
Clarke, 1989; Wright, 1991). The major product is the L-isoaspartyl, or L--aspartyl, derivative in
which the peptide bond from the aspartyl residue is made via the
-carboxyl of the side chain rather than through the
-carboxyl. This kink in the polypeptide chain, as well as the
effect of the deamidation of the asparaginyl residue, can be
detrimental to protein function.
How does an organism such as Escherichia coli cope with the inherent instability of proteins at these sites? The bacterium's high rate of protein synthesis and rapid generation time during exponential phase growth may protect it from accumulating proteins containing these isomerized residues. However, this mechanism will not suffice for nutrient-starved cells in stationary phase that are no longer dividing. E. coli, as well as a number of other organisms, can convert L-isoaspartyl residues in proteins to normal L-aspartyl residues by the action of a protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) (Fu et al., 1991). E. coli cells lacking pcm, the gene that encodes this activity, survive poorly in stationary phase or at elevated temperatures (Li and Clarke, 1992).
Repairing proteins damaged by L-isoaspartyl residue formation is an efficient way for E. coli to prevent the accumulation of abnormal proteins during stationary phase. However, there are limitations to this repair pathway (Lowenson and Clarke, 1991). First, not all damaged residues will be exposed on the protein surface accessible to the methyltransferase. Secondly, the enzyme may only recognize with high affinity a subset of the L-isoaspartyl containing damaged proteins present in the cell, allowing others to accumulate. These limitations suggest that additional mechanisms may exist for the removal of isoaspartyl residues from polypeptides, including catabolic pathways. The presence of a degradative mechanism in E. coli is supported by the existence of an isoaspartyl dipeptidase activity that has been partially purified from E. coli strain B (Haley, 1968). It was shown that the enzyme catalyzed the hydrolysis of a specific subset of L-isoaspartyl-containing dipeptides but did not catalyze the cleavage of corresponding normal aspartyl dipeptides.
Isoaspartyl
dipeptides can arise from the degradation of damaged proteins because
most proteases and peptidases do not recognize the -peptide
linkage connecting the L-isoaspartyl residue and its neighbor
on the carboxyl side (Haley et al., 1966; Murray and Clarke,
1984; Johnson and Aswad, 1990). Proteolysis of isoaspartyl-containing
polypeptides would be expected to continue until the isoaspartyl
residue is encountered, leaving an isoaspartyl dipeptide. Importantly, L-isoaspartyl residues in dipeptides are very poorly
recognized by the methyltransferase (Lowenson and Clarke, 1991).
Without a specific dipeptidase, isoaspartyl dipeptides might accumulate
in stationary phase that could be toxic to the cell or that could
partially deplete the pool of utilizable amino acids required for
viability (Mandelstam, 1958, 1960; Reeve et al., 1984a,
1984b).
In this paper, we describe the purification to homogeneity
of an E. coli isoaspartyl dipeptidase from E. coli strain MC1000 as well as from the overexpressing strain JDG100. We
show that the isolated enzyme has the greatest activity toward L-isoaspartyl dipeptides having a hydrophobic
carboxyl-terminal amino acid and exhibits no detectable activity toward
isoglutamyl, or -glutamyl, dipeptides. Sequence analysis of the
cloned gene reveals homology to the dihydroorotases and imidases rather
than to other peptidases, suggesting an evolutionary origin for the
isoaspartyl dipeptidase from these enzymes involved in pyrimidine
synthesis. In addition, mutants with a deleted isoaspartyl dipeptidase
gene were constructed and tested for their ability to survive heat
shock or extended periods in stationary phase. However, under both
conditions the dipeptidase mutants (iadA
and iadA
/pcm
)
displayed no greater loss in viability compared to their parent
strains, possibly due to the presence of a redundant isoaspartyl
dipeptidase activity.
Tryptic peptides were
generated from a concentrated solution (140 µg/ml, 500 µl) of
the Phenyl-Sepharose purified enzyme from the overexpressing strain
JDG100. The enzyme, originally 25 ml, was concentrated in a
Centriprep-10 device while exchanging the buffer to 20 mM Tris-HCl, pH 8.1. Ammonium bicarbonate (pH 8) was added to the
concentrated protein solution to 100 mM and then mixed with
urea to a final concentration of 8 M. The solution was
incubated at 23 °C for 2 hours before diluting the urea
concentration to 2 M. Trypsin (0.01 mol/mol dipeptidase) was
then added, and the reaction continued overnight at 37 °C. The
resulting peptide fragments were separated by HPLC on a reverse-phase
C18 column (2-mm diameter 30-cm length) equilibrated with
solvent A (0.1% (w/v) trifluoroacetic acid in water). The peptides were
eluted using a linear gradient (0-70%) of solvent B (0.1%
trifluoroacetic acid, 99% acetonitrile, 0.9% water (w/v/v)) over 90 min
and collected for Edman sequencing.
To confirm the results from the Takara membrane, E. coli genomic DNA was isolated from strain JA200 (Clarke and Carbon, 1976) and exhaustively digested with BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, or PvuII for Southern analysis. After separation on a 0.6% agarose gel, the DNA was transferred to an Immobilon-N membrane (Millipore) and probed as above, using the hybridization and washing conditions supplied by Millipore.
The disruption of the isoaspartyl
dipeptidase gene was accomplished by the replacement of the E. coli chromosomal locus with a DNA cassette coding for chloramphenicol
acetyltransferase, conferring chloramphenicol resistance
(Cam), driven by the pBR322 Tet
gene promoter
(a gift from R. Lloyd). A 1.5-kbp region from pJDG100, containing 246
bp upstream and 79 bp downstream of the dipeptidase gene, was
PCR-amplified (Scharf, 1990) and ligated into the unique BamHI
site in pBluescriptII SK+ (Stratagene) using the two engineered BamHI sites at either end of the 1.5-kbp PCR product,
generating pIAD001. A 757-bp fragment of the gene was removed by
digestion with EcoNI, leaving only 33% of the coding region
intact. The remainder of pIAD001 was blunt-ended with Klenow large
fragment (Sambrook et al., 1989) and ligated with the 1.5-kbp
Cam
cassette to create the deletion plasmid p
IAD, and
its orientation was determined by restriction mapping. The disruption
construct (ApaI-SpeI, both present in the
multicloning site of pBluescript) was then moved to the pBIP3 vector (a
gift from R. Maurer) that encodes both kanamycin resistance
(Km
) and sucrose sensitivity (Suc
) (Slater and
Maurer, 1993), creating the final deletion plasmid pBIP3
IAD.
Replacement of the chromosomal locus with the
iadA(
EcoNIEcoNI)::Cam
construct was done by transforming competent (Chung et
al., 1989) JC7623, a strain capable of incorporating DNA into the
chromosome by homologous recombination (Winans et al., 1985),
with pBIP3
IAD. Chromosomal insertion and the subsequent loss of
the vector in this recipient strain was positively confirmed by the
loss of Km
, Suc
, and the retention of
Cam
, creating strain JDG7623. Subsequently, the general
P1vir protocol described by Silhavy et al.(1984) was
used to transduce the deleted iadA chromosomal locus from
JDG7623 into both MC1000 and CL1010, resulting in JDG11000 and
JDG11010, respectively.
Southern analysis was done to confirm the
isoaspartyl dipeptidase gene disruption and the integrity of the CL1010
background. Genomic DNA from MC1000, CL1010, and a single putative
mutant from each background was digested with BamHI, HindIII, and PvuII. After separation on a 0.6%
agarose gel, the DNA was transferred onto an Immobilon-N membrane
(Millipore). Using the conditions supplied by Millipore, the blot was
probed with 3 different 20-mers: IAD-11 (5`-AATCACGCAGACCGTAATGA),
located just upstream from the 5` end of the isoaspartyl dipeptidase
gene; CAT-1 (5`-GACCGTTCAGCTGGATATTA), a highly conserved region within
chloramphenicol acetyltransferases; and KAN-1
(5`-GAAAGTATCCATCATGGCTG), a sequence found in neo (Km), that was used to generate the pcm deletion (Li and Clarke, 1992).
Figure 1:
Purification of the
isoaspartyl dipeptidase to homogeneity from E. coli strain
MC1000. Lane 1, an aliquot of the Phenyl-Sepharose purified
active pool (300 µl; Table 2) isolated from MC1000, as
described under ``Experimental Procedures,'' was analyzed by
SDS-PAGE and silver staining. This material was incubated overnight at
4 °C with 30 µg of insulin and an equal volume of 25% (w/v)
trichloroacetic acid. Prior to loading, the protein was pelleted in a
microcentrifuge for 20 min at 16,000 g and resuspended
in 20 µl of water. Samples were mixed 1:1 with 2
sample
buffer (Sambrook et al., 1989) and heated at 100 °C for 3
min before loading onto slab gels using the buffer system described by
Laemmli(1970). All samples were electrophoresed through an 8%
acrylamide, 0.28% (w/v) N,N-methylenebisacrylamide matrix by
the application of 20-mA constant current. On the left,
molecular size standards include phosphorylase b (97.4 kDa),
bovine serum albumin (66.2 kDa), egg white albumin (42.7 kDa), carbonic
anhydrase (31.0 kDa), and soybean trypsin inhibitor (21.5
kDa).
Figure 2:
DNA
sequence of the 98-min region of the E. coli chromosome
containing the isoaspartyl dipeptidase gene (iadA). Both
strands of a 2604-bp region of DNA encompassing the isoaspartyl
dipeptidase gene were sequenced as described under ``Experimental
Procedures.'' The template, plasmid pJDG100, was derived from
Kohara phage clone 667 DNA, which is based on the genome of E. coli strain W3110 (Kohara et al., 1987). Data from
the amino-terminal amino acid sequence analysis of the purified
isoaspartyl dipeptidase are boxed. Tryptic peptide fragments
generated from the purified dipeptidase, identified by either direct
sequencing (thin underline) or amino acid composition analysis (thick underline), are also shown. Features of this gene
include the presence of two putative sigma-70 promoter sequences (-10 and -35 regions shown, solid and dotted overline) and a possible ribosomal binding
site (rbs iadA). The open reading frame for this gene (1173
bp) encodes a predicted 41-kDa protein consisting of 390 amino acids. Arrows indicate position and direction of the primers used to
PCR-amplify the gene for the deletion construction. Dots indicate the position of bases changed to create novel BamHI sites (GGATCC). The two EcoNI sites used to
delete the coding region for the disruption are also shown. In addition
to the isoaspartyl dipeptidase gene, two other open reading frames were
found, labeled orf1 and orf2; both also have 5`
putative features. Initiator methionines for the three open reading
frames are circled.
Figure 3:
Localization of the isoaspartyl
dipeptidase gene (iadA) to the 98-min region of the E.
coli chromosome. The upper panel shows the positions of
the ordered phage clones 666-668 with respect to the base pair
numbering and restriction enzyme sites as described in EcoMap6 and
displayed using GeneScape v2.01 (K. Rudd, National Institutes of
Health, personal communication; cf. Rudd, 1992). The genomic
restriction fragments identified by Southern analysis using the 5`
probe IAD- (Probe) are shaded. The position and
transcriptional orientation of the dipeptidase gene is shown by the arrow. In the lower panel, the expected sizes of the
shaded restriction fragments from the upper panel are compared to the
observed sizes of the fragments as determined from a 0.6% agarose
gel.
The region of DNA containing the isoaspartyl dipeptidase gene was
subcloned for DNA sequencing and protein overexpression from
phage clone 667. A 5.8-kbp KpnI fragment (Fig. 3) was
ligated into the corresponding KpnI site within the
multicloning site of the pUC19 vector generating the plasmid pJDG100.
DNA sequence of a portion of the plasmid insert revealed three open
reading frames (Fig. 2). The central open reading frame
(nucleotides 1017-2190) encodes a 41-kDa protein. The deduced
amino-terminal sequence of this translated open reading frame is
identical with the amino-terminal protein sequence obtained from the
purified isoaspartyl dipeptidase. We also found that the sequence of 12
tryptic peptides generated from the isoaspartyl dipeptidase purified
from strain JDG100 (see below) were identical with those encoded by the
open reading frame (Fig. 2).
Two putative sigma-70 promoter sequences exist 70 and 83 bp upstream from the initial methionine codon of the isoaspartyl dipeptidase gene (Fig. 2). Both pairs of -35 and -10 hexanucleotide regions agree well with the consensus sequences (-35, TTGACA; and -10, TATAAT) and the 16- or 17-bp inter-region spacing is conserved among 92% of known E. coli promoter sequences (Harley and Reynolds, 1987). A very good match to the ribosomal binding sequence (GGAGTT; consensus GGAGGT) also appears 3 bp upstream from this gene (Shine and Dalgarno, 1974). The gene encodes a polypeptide of 390 amino acids, with a calculated pI of 5.02 (Protean, DNAStar). The protein has no apparent signal sequence nor membrane-spanning regions, consistent with the cytosolic location of the enzyme.
Figure 4:
Protein
sequence identity between the isoaspartyl dipeptidase from E.
coli, dihydroorotases from B. caldolyticus (Ghim et
al., 1994) and B. subtilis (Quinn et al., 1991),
and the D-hydantoinase from P. putida (LaPointe et al., 1994). Residues identical with the isoaspartyl
dipeptidase are boxed. Overall, the sequence similarity of all
three enzymes to the isoaspartyl dipeptidase, calculated using the
formula: similarity (i,j) = ((100 sum of
matches)/(length - gap residues i - gap residues j)), is approximately 13%, but this increases to 24% if only
the first 100 amino acids are considered (shaded and boxed). The four protein sequences were aligned using the
MegAlign program (DNAStar).
Figure 5: Overall similarity between the reactions catalyzed by the isoaspartyl dipeptidase, dihydroorotases, and imidases. The reversible dihydroorotase and imidase reactions are presented in the direction of their hydrolytic reaction (top to bottom) to more clearly show the resemblance to the dipeptidase reaction.
Figure 6:
Chromatographic purification of the E.
coli isoaspartyl dipeptidase from the overexpressing strain
JDG100. Cytosol from 19.2 g of DH5 cells harboring the plasmid
pJDG100 was obtained, and the isoaspartyl dipeptidase was purified as
described under ``Experimental Procedures.'' Column effluents
from the three chromatographic procedures were analyzed for protein
concentration by absorbance at 280 nm (filled circles) and
isoaspartyl dipeptidase activity (open circles). The elution
position of the dipeptidase activity from each column in this
overexpression purification was identical with that seen in the initial
purification from MC1000 (Table 2). Panel A, Sephadex
G-200 gel filtration chromatography. Only one gel filtration run was
required for this purification because the entire sample from the
ammonium sulfate step (59 ml) was concentrated to a final volume of 11
ml in a Centriprep-10 device prior to loading. The enzyme activity was
determined from 90 µl of each column fraction using the
cadmium/ninhydrin method (assay 1, see ``Experimental
Procedures'') shown as the absorbance at 505 nm. Panel B,
DEAE-cellulose chromatography. A DE52 ion exchange column was eluted
with a linear sodium chloride gradient (0-0.4 M) (solid line). Determination of the dipeptidase activity
(assaying 40 µl from each column fraction) was done as in panel
A. Panel C, Phenyl-Sepharose chromatography. Due to the high salt
concentration in these fractions, the ninhydrin/hydrindantin method
(assay 2, see ``Experimental Procedures'') was used to
analyze 90 µl of each fraction for isoaspartyl dipeptidase
activity, shown as the absorbance at 570
nm.
The similarity in the deduced amino acid sequence
to bacterial dihydroorotases (Fig. 4) prompted us to determine
whether the isoaspartyl dipeptidase could catalyze the formation of
dihydroorotate from N-carbamyl-aspartate. The structures of
the substrates for the isoaspartyl dipeptidase and the dihydroorotase
are similar, and the intramolecular cyclization reaction of
dihydroorotate formation is essentially the reverse reaction of the
amide hydrolysis catalyzed by the dipeptidase (Fig. 5). In fact,
above pH 7.1, dihydroorotases will catalyze the hydrolysis of
dihydroorotate to N-carbamyl-aspartate (Christopherson and
Jones, 1979). We found, however, that the purified dipeptidase had less
than 0.8% the activity expected of the E. coli dihydroorotase
(Washabaugh and Collins, 1984). Another similar reaction, the
hydrolysis of dihydrouracil to N-carbamyl--alanine, is
catalyzed by the imidase from rat liver (Yang et al., 1993) (Fig. 5). Although the imidases have a very broad substrate
specificity, the best substrate for the rat liver enzyme is phthalimide
(Yang et al., 1993). We therefore tested whether the purified
dipeptidase from JDG100 could catalyze the hydrolysis of this
substrate. We found that the dipeptidase had less than 0.5% of the
expected activity of this enzyme (Yang et al., 1993). Our
data, taken together with that of Haley(1968), suggest that the
dipeptidase acts primarily on a subset of isoaspartyl dipeptides.
Figure 8: Organization of the wild-type isoaspartyl dipeptidase gene locus and the chromosomal deletion construct. The position of the isoaspartyl dipeptidase gene at 98 min on the E. coli chromosome is shown in relation to the Kohara restriction enzyme sites (identified by sequence analysis), and its direction of transcription is indicated by the arrow. The location of the gene on the chromosome in kbp (boldfaced) was determined using the positional numbering established by K. Rudd in EcoMap6 (see Fig. 3). In addition, a BglI site previously unidentified in the chromosome map is boxed. This additional restriction site explains why the BglI fragment in Fig. 3is 0.5 kbp too small. The EcoNI sites used to create the null dipeptidase mutant are shown, as well as the transcriptional orientation of the inserted 1.5-kb chloramphenicol cassette. Large arrowheads bracket the region PCR-amplified from plasmid pJDG100 for the deletion construct.
We confirmed the identity of the isoaspartyl dipeptidase null mutants and the CL1010 background by Southern analysis (see ``Experimental Procedures''; data not shown). In addition, we analyzed lysates from MC1000 and JDG11000 for residual isoaspartyl dipeptidase activity by separating crude cytosol from each strain by gel filtration (Fig. 9). The mutant strain has 31% of the activity of the parent strain, and the peak of activity elutes at a lower native molecular weight. It thus appears that a secondary isoaspartyl dipeptidase activity is present that is not catalyzed by the iadA gene product.
Figure 9:
Determination of residual isoaspartyl
dipeptidase activity in cytosol from the deletion mutant JDG11000 using
gel filtration chromatography. 2 ml of crude cytosol from MC1000 (open squares) and JDG11000 (filled circles)
(isolated using steps 1-2 of the purification, see
``Experimental Procedures'') were incubated with 100 µg
of RNaseA and DNaseI at 37 °C for 50 min. Each solution was
concentrated in a Centricon-30 device (Amicon) to a final volume of 350
µl and loaded separately onto a Sephacryl-200HR (Pharmacia) column
(2-cm diameter 13-cm height, 40 ml) at room temperature. The
column was equilibrated and eluted at 23 °C with 50 mM Tris-HCl (pH 8.0) at a flow rate of 22 ml/h. Aliquots of the
1.2-ml fractions were assayed for hydrolytic activity toward the L-isoaspartyl-L-leucine dipeptide by the
cadmium/ninhydrin method (assay 1, see ``Experimental
Procedures'').
Both mutant strains, JDG11000 and JDG11010, are viable and have growth rates similar to those of their parent strains in LB and minimal M9-glucose media (data not shown).
Wild-type E. coli cells in stationary phase demonstrate an increased resistance to heat shock when compared to their response during log phase growth (Jenkins et al., 1988). The L-isoaspartyl methyltransferase is known to affect the ability of E. coli to survive a 55 °C heat shock: mutants lacking this activity undergo a rapid loss of viability after a temperature upshift (Li and Clarke, 1992). We examined the ability of the dipeptidase mutants to survive heat shock once they had reached stationary phase. After 12 min at 55 °C, 8% of the initial control cells, MC1000, were able to form colonies on LB-agar plates. Similarly, 11% of the isoaspartyl dipeptidase mutant cells remained viable after 12 min. The double mutant had only 0.1% remaining after 8 min, close to the 0.4% seen for the pcm mutant after 8 min.
To assess the
catabolic role of this isoaspartyl dipeptidase in E. coli, we
examined whether the lack of IadA in the mutant cells during starvation
could cause a cytosolic accumulation of isoaspartyl-leucine or its
secretion into the media. We thus labeled iadA mutant cells
and their parent strain with L-[H]leucine for 1.25 h and then starved
them for glucose and leucine for 17 h after the protocols of Yen et
al.(1980) and Reeve et al. (1984a). To assay for the
presence of L-isoaspartyl-L-[
H]leucine,
cytosol and media from each of the strains were reacted with o-phthaldialdehyde and fractionated by HPLC. Radioactivity
present at the elution position determined for the o-phthaldialdehyde derivative of L-isoaspartyl-L-leucine was quantitated. Consistent
with the lack of a stationary phase defect in the iadA mutant
cells, we did not detect any accumulation of L-isoaspartyl-L-[
H]leucine in
either the cytosol or media from iadA
cells
(data not shown).
The open reading frame on the 3` side (orf2, nucleotides 2255-2604) is incomplete because no termination codon is found. This open reading frame has a putative sigma-70 promoter sequence (Harley and Reynolds, 1987) as well as a putative ribosomal binding site (Shine and Dalgarno, 1974). The predicted protein encoded by orf2 has strong similarity to the amino-terminal regions of CitR and CynR from B. subtilis and E. coli, respectively, that are both members of the LysR family of transcriptional regulators (Jin and Sonenshein, 1994; Sung and Fuchs, 1992). The amino terminus of proteins belonging to this family contains a well conserved 20-amino acid region believed to form a helix-turn-helix motif that mediates specific protein-DNA contacts (Henikoff et al., 1988). Using the weight matrix calculated by Dodd and Egan for determining the potential of a sequence to be a DNA-binding helix-turn-helix motif (Dodd and Egan, 1987), this open reading frame has a raw score of 1189 for residues 28-47; other family members LysR, CitR, CynR, and MetR have scores of 1864, 1567, 1541, and 867, respectively, for their binding motif.
We have purified an E. coli isoaspartyl dipeptidase
to homogeneity using L-isoaspartyl-L-leucine as a
substrate. We mapped the structural gene encoding this enzyme (iadA) to 98 min on the chromosome and determined its sequence
by DNA as well as by partial protein sequencing. The substrate
specificity of the dipeptidase suggests that it hydrolyzes only a
subset of L-isoaspartyl-containing dipeptides. Neither
-L-glutamyl-containing dipeptides nor nonpeptide
pyrimidine analogs are substrates for this enzyme. Analysis of the
cytosolic fraction of an iadA
null mutant
strain indicated that while the majority of the isoaspartyl dipeptidase
activity of cells is due to IadA, at least one additional activity is
present.
Previous work has suggested that dipeptidases in general
are essential for the utilization of exogenous peptides as amino acid
or carbon sources (Payne, 1972; Simmonds et al., 1976). We
anticipated an additional function for isoaspartyl dipeptidase
activity, the hydrolysis of endogenous isomerized aspartyl
dipeptides. These L-isoaspartyl dipeptides can arise from the
degradation of unrepaired L-isoaspartyl-containing proteins by
proteases and peptidases specific for -peptide bonds (Fig. 10). The concentration of these proteins containing L-isoaspartyl residues presumably increases during stationary
phase because the rate of protein synthesis decreases and because they
can no longer be diluted by cell division. Proteins damaged by L-isoaspartyl formation can be repaired by the L-isoaspartyl methyltransferase, but this enzyme may only
efficiently recognize a subset of the isoaspartyl residues arising in
proteins (Lowenson and Clarke, 1991) and thus may require the action of
the isoaspartyl dipeptidase to hydrolyze the abnormal peptide linkage.
Furthermore, in stationary-phase or heat-shocked cells lacking the
methyltransferase, the steady-state level of proteins containing L-isoaspartyl residues may rise even higher, increasing the
need for the dipeptidase.
Figure 10: Possible fates for proteins containing isoaspartyl residues in E. coli. Damaged proteins containing L-isoaspartyl residues may either be degraded to isoaspartyl dipeptides or repaired to species containing normal L-aspartyl residues.
However, our attempts to observe either a
stationary-phase or heat-shock survival defect in either of the
dipeptidase mutants were unsuccessful. These results are consistent
with in vivo labeling experiments using L-[H]leucine. We detected no cytosolic
accumulation or secretion into the media of L-isoaspartyl-L-leucine in iadA
cells over their parent strain after a 17-h growth period. It is
possible that the residual isoaspartyl dipeptidase activity seen in the
deletion strain (Fig. 9) is sufficient to preclude the build up
of isoaspartyl dipeptides and prevent the appearance of a phenotype
that might result from the sequestration of a fraction of the internal
amino acid pool as isoaspartyl dipeptides and/or the toxicity of
isoaspartyl dipeptides themselves (Fig. 10). In either case, the
existence of at least two proteins capable of hydrolyzing isoaspartyl
dipeptides may suggest this catabolic enzyme's importance.
Recently, the -aspartyl dipeptidase gene (pepE) from E. coli was mapped to 90 min on the chromosome (Conlin et
al., 1994), and its sequence was determined (Blattner et
al., 1993). The
-glutamyltranspeptidase (Ggt) has also been
isolated from E. coli and sequenced, and its gene has been
mapped to 76 min on the chromosome (Suzuki et al., 1993).
Although both enzymes have substrates reminiscent of those for the
isoaspartyl dipeptidase, no sequence similarity exists between PepE,
Ggt, or any other known peptidase and the isoaspartyl dipeptidase.
However, sequence similarities have been found with bacterial
dihydroorotases and imidases, enzymes involved in pyrimidine
metabolism. Amino acid identities exist throughout the protein
sequences, but are more concentrated at the amino terminus (Fig. 4), a region of the dihydroorotase suggested to be
responsible for substrate binding (Buckholz and Cooper, 1991).
Isoaspartyl dipeptides do structurally resemble dihydroorotate and
dihydrouracil (Fig. 5), substrates for the reverse reaction of
dihydroorotases and imidases, respectively. The amide bond hydrolyzed
in these three substrates is also cleaved at a similar position.
Instead of modifying an existing peptidase to accommodate a peptide
bond containing an additional methylene group, it appears that E.
coli adopted the catalytic mechanism of seemingly unrelated
metabolic enzymes to complete the task.
An isoaspartyl dipeptidase activity has also been found in rat liver and kidney (EC 3.4.19.5) (Dorer et al., 1968). This enzyme, however, has a substrate specificity distinct from that of the bacterial enzyme. For the rat enzyme, L-isoaspartyl-glycine is a much better substrate than L-isoaspartyl-L-leucine (Dorer et al., 1968), the opposite of the E. coli enzyme specificity described here and by Haley(1968). In addition, the mammalian enzyme has an exopeptidase activity that can hydrolyze isoaspartyl residues from the amino terminus of a tripeptide (Dorer et al., 1968) that the E. coli enzyme seems to lack (Haley, 1968). For this reason, we have not identified the E. coli IadA with the EC number of the mammalian enzyme, but suggest the bacterial enzyme be classified separately. The physiological function of the mammalian isoaspartyl dipeptidase has not been established (Burton et al., 1989), but it is possible that it too is involved in the metabolism of endogenous peptides resulting from the degradation of isoaspartyl-containing proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15029[GenBank].