Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gary, J. D.
Right arrow Articles by Clarke, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gary, J. D.
Right arrow Articles by Clarke, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Volume 270, Number 8, Issue of February 24, 1995 pp. 4076-4087
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of an Isoaspartyl Dipeptidase from Escherichia coli(*)

(Received for publication, June 27, 1994; and in revised form, November 28, 1994)

Jonathan D. Gary ,  Steven Clarke (§)

From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90024-1569

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-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.

INTRODUCTION

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-beta-aspartyl, derivative in which the peptide bond from the aspartyl residue is made via the beta-carboxyl of the side chain rather than through the alpha-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 beta-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.

EXPERIMENTAL PROCEDURES

Bacterial Growth

Bacteria were grown in Luria-Bertani (LB) (^1)broth (Difco) for all manipulations except for testing phenotypes in liquid culture, where M9-glucose media (Miller, 1972) was used. E. coli strain MC1000 and its derivatives described here require the addition of both L-leucine (40 µg/ml) and thiamine (1 µg/ml) to the minimal media. When appropriate, the antibiotics kanamycin sulfate, chloramphenicol, and sodium ampicillin were added to a final concentration of 80 µg/ml. Liquid cultures were grown at 37 °C in a New Brunswick Innova incubator shaker at 250 rpm, and colonies were cultured on 1.5% agar LB plates (Difco Bacto-agar) in a 37 °C incubator.

Purification of the E. coli Isoaspartyl Dipeptidase

Steps 1-6 of this procedure are derived from the partial purification of the isoaspartyl dipeptidase described by Haley(1968).

Steps 1-4: Homogenization and Initial Precipitations

1) E. coli MC1000 cells from a late logarithmic-phase culture (6 liters) grown in LB broth were pelleted at 4,500 times g for 15 min at 23 °C. The pellet (13.5 g) was then resuspended in 1/20 the original volume (300 ml) of 50 mM Tris-HCl (pH 8.1, measured at 20 °C) and repelleted at 5,000 times g for 15 min at 4 °C. The cells were washed in buffer again and resuspended in 27 ml of 50 mM Tris-HCl, pH 8.1. 2) Cells were disrupted by passage twice through a French pressure cell (American Instrument Company) at 20,000 p.s.i. Crude cytosol was isolated from the lysed cells by centrifugation at 23,000 times g for 30 min at 4 °C. 3) To the resulting supernatant (28.5 ml), 0.05 volume (1.4 ml) of 1 M manganese chloride was added, and the mixture was stirred at 0 °C for 45 min and then centrifuged at 23,000 times g as described above. 4) Solid ammonium sulfate (16.6 g) was mixed into the supernatant at 0 °C (25.4 ml) over a 1-min period to give the equivalent of 90% saturation at 25 °C. The solution was stirred for 3 h at 0 °C and centrifuged as described above. The precipitate was dissolved in a minimal amount (12 ml) of 50 mM Tris-HCl, pH 8.1, and dialyzed (cutoff 3,500 Da, Spectrapor) against 1 liter of the same buffer for 12 h at 4 °C with one change of buffer.

Step 5: Sephadex G-200 Chromatography

An aliquot of the dialyzed precipitate from step 4 (12.6 ml) was loaded at room temperature onto a Sephadex G-200 (Pharmacia Biotech Inc.) chromatography column (3-cm diameter times 38-cm height, 270 ml) equilibrated at 23 °C with 50 mM Tris-HCl, pH 8.1. The sample was loaded and eluted from the column by gravity flow at a hydrostatic head of 10 cm at a flow rate of 12.6 ml/h, and 10-min fractions were collected. The dipeptidase activity eluted with the initial 280 nm absorbing peak. Active fractions were pooled and stored at -20 °C for a maximum of 4 days before the next step.

Step 6: DEAE-Cellulose Anion Exchange Chromatography

At 4 °C, the combined Sephadex G-200 active pool from three of the above column runs (75 ml) was loaded onto a DE52 (Whatman) column (2.5-cm diameter times 7-cm height, 35 ml), pre-equilibrated at 4 °C with 20 mM Tris-HCl, pH 8.1. After loading the sample, the column was washed with 2 column volumes of the equilibration buffer and subsequently eluted with a linear sodium chloride gradient (0 to 0.4 M in the equilibration buffer, 500 ml) followed by a 3-column-volume high-salt wash (0.4 M sodium chloride in the equilibration buffer) at 4 °C. Ten-min fractions were collected at a flow rate of 13.4 ml/h, and the effluent was monitored for isoaspartyl dipeptidase activity. Activity was found to consistently elute between sodium chloride concentrations of 0.3 and 0.4 M. These active fractions were then pooled (32 ml) and concentrated in a Centriprep-10 device (Amicon) to a volume of 4.7 ml for the final chromatography step.

Step 7: Hydrophobic Interaction Chromatography

Potassium monobasic phosphate was mixed with 20 mM Tris-HCl, pH 8.1, to a final concentration of 1 M, and the pH of the solution was readjusted to 8.1 with KOH. We added this solution (3.1 ml) to the concentrated active pool from step 6 to bring the potassium phosphate concentration to 0.4 M. This sample was then loaded on to a Fast Flow Phenyl-Sepharose (Pharmacia) column (1.5-cm diameter times 12-cm height, 22 ml) equilibrated with 0.4 M potassium phosphate in 20 mM Tris-HCl, pH 8.1. The column was run at a flow rate of 15 ml/h at 4 °C, and fractions were collected every 10 min. The dipeptidase was eluted isocratically with the loading buffer and usually appeared between fractions 15 and 30.

Isoaspartyl Dipeptidase Activity Assays

In general, isoaspartyl dipeptidase was incubated with 1 mML-isoaspartyl-L-leucine (beta-Asp-Leu; Sigma; [alpha] = -24.9° (literature value = -29.6° (Buchanan et al., 1966))) in a final volume of 100 µl with 50 mM Tris-HCl, pH 8.1, in a 1.5-ml polypropylene microcentrifuge tube. We confirmed the L-configuration of the aspartyl residue by acid hydrolysis of the dipeptide and subsequent HPLC analysis as described (Brunauer and Clarke, 1986). Incubations were performed at 37 °C for 5-30 min, depending on enzyme concentration, and were stopped by the addition of 50 µl of 25% (w/v) trichloroacetic acid. To ensure that initial velocity conditions were maintained, reactions were stopped before products exceeded 15% of the initial substrate level. After centrifugation at 16,000 times g for 3 min, an aliquot of the supernatant was analyzed by one of the following procedures. Both ninhydrin-based assays take advantage of the lower absorbance of a ninhydrin dipeptide adduct compared to the product of ninhydrin and free amino acids. L-Isoaspartyl-glycine, L-isoaspartyl-L-histidine, L-isoaspartyl-L-valine, L-isoglutamyl-L-leucine, L-isoglutamyl-glycine, L-isoglutamyl-L-cysteine, and L-isoglutamyl-L-histidine dipeptides, tested as alternative substrates, were obtained from Sigma.

Assay 1: Cadmium/Ninhydrin

This procedure, used to assay fractions from steps 5 and 6 of the purification, is derived from those of Setlow(1975) and Plancot and Han(1969). A sample (30 µL) of the trichloroacetic acid-quenched hydrolysis supernatant was added to 1 ml of a 1% (w/v) ninhydrin solution containing 1 mg/ml cadmium acetate in a solvent of 85% ethanol, 15% acetic acid (v/v). The reaction mixture was then incubated at 70 °C for exactly 10 min in a screw-capped 2.0-ml polypropylene microcentrifuge tube and allowed to cool for 5 min before the absorbance at 505 nm was measured.

Assay 2: Ninhydrin/Hydrindantin

This assay was used to analyze samples in step 7 of the purification, where the high salt content of the fractions interfered with assay 1 described above, and is based on Moore(1968). A sample (30 µl) of the hydrolysis supernatant was diluted with water to a final volume of 0.7 ml in a borosilicate test tube (12 times 75 mm) and then mixed with 300 µl of a 2% (w/v) ninhydrin solution containing 3 mg/ml hydrindantin in a solvent of 75% dimethyl sulfoxide, 25% 4.0 M lithium acetate, pH 5.2 (v/v). The mixture was heated for 15 min at 100 °C and immediately plunged into an ice bath for 1 min, and the absorbance at 570 nm was measured.

Assay 3: HPLC Amino Acid Analysis

A modification of the method of Jones and Gilligan(1983) was used. An aliquot (10 µl) of the hydrolysis supernatant was diluted into 657 µl of 0.4 M potassium borate, pH 10.4. To 10 µl of this diluted solution was added 40 µl of a 0.4% (w/v) o-phthaldialdehyde (Fluka) derivatizing solution containing 10% methanol and 0.4% beta-mercaptoethanol in 0.4 M potassium borate, pH 10.4. The reaction was allowed to proceed for 30 s before 25 µl of this solution were fractionated on a Waters Pico-Tag reverse-phase column (3.9-mm diameter times 150-mm length) equilibrated at 37 °C in solvent C (1% tetrahydrofuran, 9.5% methanol, 89.5% 0.1 M sodium acetate, pH 7.22 (v/v/v)). The derivatized products were eluted at 37 °C using the following gradient at a flow rate of 1.0 ml/min: 0-1 min, 0-16% solvent D (100% methanol); 1-14 min, isocratic with 16% solvent D; 14-19 min, 16-36% solvent D; 19-24 min, isocratic with 36% solvent D; 24-31 min, 36-56% solvent D; 31-37 min, isocratic with 56% solvent D; 37-38 min, 56-100% solvent D; 38-43 min, isocratic with 100% solvent D; 43-46 min, 100-0% solvent D. The column was equilibrated for 15 min with solvent C prior to the next injection. The extent of L-isoaspartyl-L-leucine hydrolysis was quantitated by averaging the amount of free aspartate and leucine formed during the incubation based on the fluorescence of amino acid standards (50 pmol). Fluorescence was detected using a Gilson (model 121) fluorometer with an excitation wavelength of 305-395 nm and an emission wavelength of 430-470 nm. Substrate-only or enzyme-only blanks were used as controls.

Protein Determination

A modification of the Lowry procedure (Bailey, 1967) was used to determine the concentration of protein after precipitation with 1 ml of 10% (w/v) trichloroacetic acid. Bovine serum albumin was used as a standard. Alternatively, protein concentration was approximated by absorbance at 280 nm (1 A unit equivalent to 1 mg/ml) or at 230 nm (1 A unit equivalent to 0.2 mg/ml).

Amino Acid Sequencing

Amino acid sequence analysis of the amino terminus and tryptic peptide fragments of the purified enzyme was performed by Dr. Audree Fowler at the UCLA Protein Microsequencing Facility with a Porton 2090E gas-phase sequencer with on-line HPLC detection. Amino-terminal sequence analysis was obtained from approximately 45 µg of the Phenyl-Sepharose purified enzyme from MC1000. The isoaspartyl dipeptidase (31.5 ml) was concentrated in a Centriprep-10 device (Amicon), and the buffer was exchanged to 20 mM Tris-HCl, pH 8.1. The final volume (4.4 ml) was then lyophilized to dryness and resuspended in 150 µl of 2 times sample buffer (Sambrook et al., 1989) and separated by SDS-PAGE. The proteins were subsequently electroblotted (40 min at 70 V) onto a polyvinylidene difluoride membrane in 25 mM Tris base, 10 mM glycine, 0.5 mM dithiothreitol, in 10% methanol, 90% water (v/v) at pH 9 and Coomassie-stained to locate the band corresponding to the isoaspartyl dipeptidase. The excised band was then subjected to automated Edman sequencing.

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 times 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.

Oligonucleotide Synthesis

Oligonucleotides were synthesized on a Gene Assembler Plus DNA synthesizer (Pharmacia Biotech) using beta-cyanoethyl N,N-diisopropylphosphoramidite chemistry on a 0.2 µmol scale. The DNA was hydrolyzed from the solid support by incubation in 1 ml of ammonium hydroxide for 15 h at 55 °C (Reynolds and Buck, 1992) and precipitated from the solution using the standard sodium acetate method (Sambrook et al., 1989).

Chromosomal Mapping of the Isoaspartyl Dipeptidase Gene

Initial mapping of the isoaspartyl dipeptidase gene was accomplished by probing the E. coli Gene Mapping Membrane (Takara Biochemical Inc.) with a 5`-P-labeled degenerate oligonucleotide probe (30 pmol, 4 times 10^6 cpm/pmol) based on the amino-terminal protein sequence results. Probe IAD-alpha, corresponding to the amino acid sequence MIDYTAA, was synthesized as a fully degenerate (192-fold) 20-mer. The hybridization and washing conditions suggested by the membrane manufacturer were used.

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.

DNA Sequencing

The isoaspartyl dipeptidase gene as well as flanking regions were sequenced by the dideoxy chain-terminating method (Tabor and Richardson, 1989) using the Sequenase Version 2.0 kit (United States Biochemical) and alpha-[S]dATP incorporation (DuPont NEN). The walking primer method was used with the pJDG100 template. The sequence described in the text was determined from both strands and with the incorporation of dideoxyinosine.

Cloning and Disruption of the Isoaspartyl Dipeptidase Gene

DNA from the Kohara-ordered phage clone 667 (Kohara et al., 1987) (a gift from M. Leonard and W. Wickner) was isolated (Sambrook et al., 1989) and subsequently digested with KpnI. The 5.8-kbp KpnI fragment containing the amino-terminal region of the isoaspartyl dipeptidase gene by Southern analysis, was ligated into the corresponding KpnI site within the multicloning site of the pUC19 vector (Life Technologies, Inc.) such that iadA transcription is in the same direction as lacZ, generating pJDG100.

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^r), driven by the pBR322 Tet^r 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^r cassette to create the deletion plasmid pDeltaIAD, 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^r) and sucrose sensitivity (Suc^s) (Slater and Maurer, 1993), creating the final deletion plasmid pBIP3DeltaIAD.

Replacement of the chromosomal locus with the DeltaiadA(DeltaEcoNIEcoNI)::Cam^r 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 pBIP3DeltaIAD. Chromosomal insertion and the subsequent loss of the vector in this recipient strain was positively confirmed by the loss of Km^r, Suc^s, and the retention of Cam^r, 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^r), that was used to generate the pcm deletion (Li and Clarke, 1992).

Long-term Stationary Phase and Heat Shock Survival

Strains MC1000, CL1010, JDG11000, and JDG11010 were grown for 10 days in M9-glucose media (without antibiotics, 40 µg/ml L-leucine, and 1 µg/ml thiamine) as described by Li and Clarke(1992). The viable cell number was determined every 2 days by plating dilutions onto LB-agar plates. Strains were also grown in M9-glucose media as described above for 24 h before the 55 °C heat challenge as described by Li and Clarke(1992). Aliquots were removed from the culture at 55 °C every 2 min, and the number of cells remaining was determined by plating onto LB-agar plates. Each strain was analyzed in at least two replicates for each experiment.

RESULTS

Purification of the Isoaspartyl Dipeptidase from E. coli Strain MC1000

We purified the dipeptidase to homogeneity from strain MC1000 (Table 1) as described under ``Experimental Procedures.'' SDS-PAGE analysis of the combined fractions containing isoaspartyl dipeptidase activity eluting from the final Phenyl-Sepharose column revealed a single polypeptide band at 41 kDa (Fig. 1). Our purification resulted in at least a 3000-fold enrichment of the dipeptidase specific activity over crude cytosol (Table 2).

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 times g and resuspended in 20 µl of water. Samples were mixed 1:1 with 2 times 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).


Mapping and Sequencing of the Isoaspartyl Dipeptidase Gene

The-41 kDa polypeptide purified from strain MC1000 ( Fig. 1and Table 2) was subjected to automated Edman sequencing as described under ``Experimental Procedures.'' The 24 identified amino-terminal residues (Fig. 2) showed no homology with any previously sequenced protein or translated DNA using the BLAST-protein searching algorithm (BLASTP) at the National Center for Biotechnology Information (queried 12/92). We then used a radiolabeled degenerate oligonucleotide corresponding to the amino-terminal protein sequence to map the isoaspartyl dipeptidase gene to 98 min on the E. coli chromosome at a position about 4589.6 kbp from the thrA gene at 0 min (Fig. 3). Mapping was initially accomplished using a membrane preblotted with 476 overlapping phage clones encompassing 99% of the E. coli genome (Kohara et al., 1987). Autoradiography revealed phage clone 667 as the only positive. We confirmed the mapping results by Southern analysis of genomic DNA restricted with the eight enzymes used to generate the Kohara map (Kohara et al., 1987) (Fig. 3).

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-alpha (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.

Sequence Similarities between the Isoaspartyl Dipeptidase and Other Proteins

The results of a BLASTP search (12/93) with the entire translated isoaspartyl dipeptidase gene revealed no sequence similarity to other peptidases or proteases. However, significant similarities were found to bacterial dihydroorotases (EC 3.5.2.3), as well as to the functionally related imidases (EC 3.5.2.2). These enzymes are involved in the synthesis and degradation of pyrimidines. The closest matches are seen with the Bacillus subtilis and Bacillus caldolyticus dihydroorotases and the Pseudomonas putidaD-hydantoinase, an imidase (Fig. 4). The highest level of similarity is located in the amino-terminal 100 residues, where an overall identity of about 24% exists between the isoaspartyl dipeptidase and the other enzymes. Interestingly, the substrates for the three enzymes have a similar structural geometry, and hydrolysis occurs adjacent to similar functionalities (Fig. 5). These data suggest that the three types of enzymes have a structure/function relationship and are evolutionarily related. In addition, a potential zinc-binding motif is found at position 62-71 (PGFIDQHVHL) of the dipeptidase; work done with the E. coli dihydroorotase suggests that the two conserved histidines in this sequence may be ligands for a catalytic zinc ion (Washabaugh and Collins, 1986; Brown and Collins, 1991).

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 times 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.


Purification of the Isoaspartyl Dipeptidase from an Overexpressing Strain JDG100

To obtain enough material for activity studies, the isoaspartyl dipeptidase was purified from strain JDG100 ( Fig. 6and 7), which overexpresses the enzyme at least 40-fold over MC1000. This strain harbors the multicopy plasmid containing the 5.8-kbp KpnI chromosomal fragment encompassing the dipeptidase gene. The specific activity of the enzyme purified from JDG100 was 19 µmol/min/mg using the L-isoaspartyl-L-leucine substrate.

Figure 6: Chromatographic purification of the E. coli isoaspartyl dipeptidase from the overexpressing strain JDG100. Cytosol from 19.2 g of DH5alpha 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.


Enzyme Specificity of the Isoaspartyl Dipeptidase

Kinetic analysis using the purified isoaspartyl dipeptidase from strain MC1000 confirmed the previously determined K(m) value of 0.8 mM for the L-isoaspartyl-L-leucine substrate (data not shown) (Haley, 1968). To further examine the substrate specificity of the isoaspartyl dipeptidase, we incubated the Phenyl-Sepharose-purified enzyme from JDG100 with four -glutamyl dipeptides: either 1 mM -L-glutamyl-L-leucine, -L-glutamyl-glycine, -L-glutamyl-L-cysteine, or -L-glutamyl-L-histidine. No hydrolysis was detected using any of these substrates under conditions where as little as 10% of the control activity (L-isoaspartyl-L-leucine) is readily detected (data not shown). Furthermore, the dipeptidase had little or no observable activity toward L-isoaspartyl-glycine or L-isoaspartyl-L-histidine, but did show significant activity toward L-isoaspartyl-L-valine (data not shown) (cf. Haley, 1968). These results indicate that the isoaspartyl dipeptidase does not appear to be involved in glutathione metabolism and does not have overlapping activity with the -glutamyltranspeptidase (EC 2.3.2.2) but does hydrolyze L-isoaspartyl dipeptides with hydrophobic amino acids at the carboxyl terminus.

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-beta-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.

Disruption of the Chromosomal Isoaspartyl Dipeptidase Gene

We created two deletion strains by the replacement of 65% of the amino acid coding region of the isoaspartyl dipeptidase gene with a chloramphenicol resistance cassette (Fig. 8). Incorporation of the deletion construct into the E. coli chromosome was accomplished in strain JC7623, and the deletion locus was moved into the desired backgrounds using general transduction mediated by P1vir phage as described under ``Experimental Procedures.'' Our use of the pBIP3 vector to transform JC7623 aided greatly in the positive selection for strain JDG7623, containing the genomically integrated iadA deletion construct, because the sacB gene contained on pBIP3 confers sucrose sensitivity on cells harboring the plasmid. By plating transformants onto LB-agar supplemented with 5% (w/v) sucrose and chloramphenicol, only cells that had undergone homologous recombination and subsequent elimination of the plasmid grew at 30 °C. In addition, the kanamycin resistance marker, also present in pBIP3, was lost upon plasmid elimination. The gene replacement was then carried out in both MC1000 (parent) and CL1010 (pcm mutant) backgrounds using the P1vir phage lysate generated from JDG7623; the resulting strains were designated JDG11000 and JDG11010, respectively.

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 times 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).

Effects of a Disrupted Isoaspartyl Dipeptidase Gene on E. coli While in Stationary Phase

The apparent deleterious effects of isoaspartyl residues within polypeptides upon long-term stationary phase survival of E. coli in minimal media is observed when the protein responsible for repairing these abnormal residues, Pcm, is absent (Li and Clarke, 1992). Because the isoaspartyl dipeptidase is also responsible for removing isoaspartyl residues, albeit from dipeptides, we tested the dipeptidase null mutant and a dipeptidase/methyltransferase double mutant for a similar survival phenotype in minimal media. After 10 days in stationary phase, 36% of the original iadA cells (JDG11000) are still able to form colonies compared to 32% for the pcm/iadA strain MC1000. The pcm strain CL1010 has a more dramatic loss in viability; only 4% of the original cell number can form colonies on day 10. The phenotype of JDG11010 exhibited a survival rate similar to its parent strain, 5% surviving on day 10.

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-[^3H]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-[^3H]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-[^3H]leucine in either the cytosol or media from iadA cells (data not shown).

Protein Sequence Features of Orf1 and Orf2

Two additional open reading frames were found adjacent to the isoaspartyl dipeptidase gene and are transcribed in the same direction (Fig. 3). The open reading frame on the 5` side (orf1, nucleotides 544-1005) has a putative ribosomal binding site (Shine and Dalgarno, 1974) but no clearly definable sigma-70 promoter (Harley and Reynolds, 1987), and potentially encodes a 16-kDa protein (153 amino acids). No substantial similarity was found between this sequence and those in the database at the National Center for Biotechnology Information. The sequence itself, however, does contain some interesting features. The sequence TXLL is repeated four times at positions 26, 45, 70, and 101. In addition, the regions flanking the repeats at positions 70 and 101 are very similar.

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.

DISCUSSION

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 alpha-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-[^3H]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 alpha-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.

FOOTNOTES

*
This work was supported by Grant MCB-9305405 from the National Science Foundation, by Grant GM26020 from the National Institutes of Health, and by United States Public Health Service Training Grant GM-07185 (to J. D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15029[GenBank].

§
To whom correspondence and reprint requests should be addressed. Tel.: 310-825-8754; Fax: 310-206-7286; clarke{at}ewald.mbi.ucla.edu.

(^1)
The abbreviations used are: LB, Luria-Bertani; kbp, kilobase pair(s); HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We thank Audree Fowler (UCLA Protein Microsequencing Facility) for her expert amino acid sequencing work, Marilyn Leonard and William Wickner (Dartmouth University) for their gift of the phage clone 667, Russell Maurer (Case Western Reserve University) for the gift of the pBIP3 plasmid, Kenneth Rudd (National Institutes of Health) for helpful advice with EcoMap6, and Robert Lloyd (University of California, Los Angeles) for donating the chloramphenicol cassette.

REFERENCES

  1. Bailey, J. L. (1967) in Techniques in Protein Chemistry , 2nd ed, pp. 340-341, Elsevier Science Publishers B.V., Amsterdam
  2. Blattner, F. R., Burland, V., Plunkett, G., Sofia, H. J., and Daniels, D. L. (1993) Nucleic Acids Res. 21, 5408-5417 [Abstract/Free Full Text]
  3. Brown, D. C., and Collins, K. D. (1991) J. Biol. Chem. 266, 1597-1604 [Abstract/Free Full Text]
  4. Brunauer, L. S., and Clarke, S. (1986) J. Biol. Chem. 261, 12538-12543 [Abstract/Free Full Text]
  5. Buchanan, D. L., Haley, E. E., Dorer, F. E., and Corcoran, B. J. (1966) Biochemistry 5, 3240-3245 [CrossRef][Medline] [Order article via Infotrieve]
  6. Buckholz, R. G., and Cooper, T. G. (1991) Yeast 7, 913-923 [CrossRef][Medline] [Order article via Infotrieve]
  7. Burton, E. G., Schoenhard, G. L., Hill, J. A., Schmidt, R. E., Hribar, J. D., Kotsonis, F. N., and Oppermann, J. A. (1989) J. Nutr. 119, 713-721
  8. Casadaban, M. J., and Cohen, S. N. (1980) J. Mol. Biol. 138, 179-207 [CrossRef][Medline] [Order article via Infotrieve]
  9. Christopherson, R. I., and Jones, M. E. (1979) J. Biol. Chem. 254, 12506-12512 [Free Full Text]
  10. Chung, C. T., Niemela, S. L., and Miller, R. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2172-2175 [Abstract/Free Full Text]
  11. Clarke, L., and Carbon, J. (1976) Cell 9, 91-99 [CrossRef][Medline] [Order article via Infotrieve]
  12. Clarke, S., Stephenson, R. C., and Lowenson, J. D. (1992) in Stability of Protein Pharmaceuticals (Ahern, T. J., and Manning, M. C., eds) Part A, pp. 1-29, Plenum Publishing Co., New York _
  13. Conlin, C. A., Knox, T. M., and Miller, C. G. (1994) J. Bacteriol. 176, 1552-1553 [Free Full Text]
  14. Dodd, I. B., and Egan, J. B. (1987) J. Mol. Biol. 194, 557-564 [CrossRef][Medline] [Order article via Infotrieve]
  15. Dorer, F. E., Haley, E. E., and Buchanan, D. L. (1968) Arch. Biochem. Biophys. 127, 490-495 [CrossRef][Medline] [Order article via Infotrieve]
  16. Fu, J. C., Ding, L., and Clarke, S. (1991) J. Biol. Chem. 266, 14562-14572 [Abstract/Free Full Text]
  17. Ghim, S. Y., Nielsen, P., and Neuhard, J. (1994) Microbiology 140, 479-491 [Abstract/Free Full Text]
  18. Haley, E. E. (1968) J. Biol. Chem. 243, 5748-5752 [Abstract/Free Full Text]
  19. Haley, E. E., Corcoran, B. J., Dorer, F. E., and Buchanan, D. L. (1966) Biochemistry 5, 3229-3235 [CrossRef][Medline] [Order article via Infotrieve]
  20. Harley, C. B., and Reynolds, R. P. (1987) Nucleic Acids Res. 15, 2343-2361 [Abstract/Free Full Text]
  21. Henikoff, S., Haughn, G. W., Calvo, J. M., and Wallace, J. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606 [Abstract/Free Full Text]
  22. Jenkins, D. E., Schultz, J. E., and Matin, A. (1988) J. Bacteriol. 170, 3910-3914 [Abstract/Free Full Text]
  23. Jin, S., and Sonenshein, A. L. (1994) J. Bacteriol. 176, 4669-4679 [Abstract/Free Full Text]
  24. Johnson, B. A., and Aswad, D. W. (1990) Biochemistry 29, 4373-4380 [CrossRef][Medline] [Order article via Infotrieve]
  25. Jones, B. N., and Gilligan, J. P. (1983) J. Chromatogr. 266, 471-482 [CrossRef][Medline] [Order article via Infotrieve]
  26. Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508 [CrossRef][Medline] [Order article via Infotrieve]
  27. Laemmli, U. K. (1970) Nature 227, 680-685 [CrossRef][Medline] [Order article via Infotrieve]
  28. LaPointe, G., Viau, S., LeBlanc, D., Robert, N., and Morin, A. (1994) Appl. Environ. Microbiol. 60, 888-895 [Abstract/Free Full Text]
  29. Li, C., and Clarke, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9885-9889 [Abstract/Free Full Text]
  30. Lowenson, J. D., and Clarke, S. (1991) J. Biol. Chem. 266, 19396-19406 [Abstract/Free Full Text]
  31. Mandelstam, J. (1958) Biochem. J. 69, 110-119 [Medline] [Order article via Infotrieve]
  32. Mandelstam, J. (1960) Bacteriol. Rev. 24, 289-308 [Free Full Text]
  33. Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  34. Moore, S. (1968) J. Biol. Chem. 243, 6281-6283 [Abstract/Free Full Text]
  35. Murray, E. D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732 [Abstract/Free Full Text]
  36. Payne, J. W. (1972) J. Gen. Microbiol. 71, 267-279 [Abstract/Free Full Text]
  37. Plancot, M. T., and Han, K. (1969) Bull. Soc. Chim. Biol. (Paris) 51, 1111-1114 [Medline] [Order article via Infotrieve]
  38. Quinn, C. L., Stephenson, B. T., and Switzer, R. L. (1991) J. Biol. Chem. 266, 9113-9127 [Abstract/Free Full Text]
  39. Reeve, C. A., Bockman, A. T., and Matin, A. (1984a) J. Bacteriol. 157, 758-763 [Abstract/Free Full Text]
  40. Reeve, C. A., Amy, P. S., and Matin, A. (1984b) J. Bacteriol. 160, 1041-1046 [Abstract/Free Full Text]
  41. Reynolds, T. R., and Buck, G. A. (1992) BioTechniques 12, 518-521 [Medline] [Order article via Infotrieve]
  42. Rudd, K. E. (1992) in A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Miller, J., ed) pp. 2.3-2.43, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  43. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  44. Scharf, S. J. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 84-91, Academic Press, San Diego
  45. Setlow, P. (1975) J. Bacteriol. 122, 642-649 [Abstract/Free Full Text]
  46. Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1342-1346 [Abstract/Free Full Text]
  47. Silhavy, T. J., Berman, M. L., and Enquist, L. M. (1984) Experiments with Gene Fusions , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  48. Simmonds, S., Szeto, K. S., and Fletterick, C. G. (1976) Biochemistry 15, 261-271 [CrossRef][Medline] [Order article via Infotrieve]
  49. Slater, S., and Maurer, R. (1993) J. Bacteriol. 175, 4260-4262 [Abstract/Free Full Text]
  50. Stephenson, R. C., and Clarke, S. (1989) J. Biol. Chem. 264, 6164-6170 [Abstract/Free Full Text]
  51. Sung, Y. C., and Fuchs, J. A. (1992) J. Bacteriol. 174, 3645-3650 [Abstract/Free Full Text]
  52. Suzuki, H., Hashimoto, W., and Kumagai, H. (1993) J. Bacteriol. 175, 6038-6040 [Abstract/Free Full Text]
  53. Tabor, S., and Richardson, C. C. (1989) J. Biol. Chem. 264, 6447-6458 [Abstract/Free Full Text]
  54. Washabaugh, M. W., and Collins, K. D. (1984) J. Biol. Chem. 259, 3293-3298 [Abstract/Free Full Text]
  55. Washabaugh, M. W., and Collins, K. D. (1986) J. Biol. Chem. 261, 5920-5929 [Abstract/Free Full Text]
  56. Winans, S. C., Elledge, S. J., Krueger, J. H., and Walker, G. C. (1985) J. Bacteriol. 161, 1219-1221 [Abstract/Free Full Text]
  57. Wright, H. T. (1991) Crit. Rev. Biochem. Mol. Biol. 26, 1-52 [Medline] [Order article via Infotrieve]
  58. Yang, Y. S., Ramaswamy, S., and Jakoby, W. B. (1993) J. Biol. Chem. 268, 10870-10875 [Abstract/Free Full Text]
  59. Yen, C., Green, L., and Miller, C. G. (1980) J. Mol. Biol. 143, 35-48 [CrossRef][Medline] [Order article via Infotrieve]
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Michalska, K. Brzezinski, and M. Jaskolski
Crystal Structure of Isoaspartyl Aminopeptidase in Complex with L-Aspartate
J. Biol. Chem., August 5, 2005; 280(31): 28484 - 28491.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
D. H. Broder and C. G. Miller
DapE Can Function as an Aspartyl Peptidase in the Presence of Mn2+
J. Bacteriol., August 15, 2003; 185(16): 4748 - 4754.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Frankel, N. Yadav, J. Lee, T. L. Branscombe, S. Clarke, and M. T. Bedford
The Novel Human Protein Arginine N-Methyltransferase PRMT6 Is a Nuclear Enzyme Displaying Unique Substrate Specificity
J. Biol. Chem., January 25, 2002; 277(5): 3537 - 3543.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
R. A. Larsen, T. M. Knox, and C. G. Miller
Aspartic Peptide Hydrolases in Salmonella enterica Serovar Typhimurium
J. Bacteriol., May 15, 2001; 183(10): 3089 - 3097.
[Abstract] [Full Text]

Home page
 J. Bacteriol.Home page
A. Guigueno, J. Dassa, P. Belin, and P. L. Boquet
Oversynthesis of a New Escherichia coli Small RNA Suppresses Export Toxicity of DsbA'-PhoA Unfoldable Periplasmic Proteins
J. Bacteriol., February 15, 2001; 183(4): 1147 - 1158.
[Abstract] [Full Text]

Home page
 J. Bacteriol.Home page
A. Toguchi, M. Siano, M. Burkart, and R. M. Harshey
Genetics of Swarming Motility in Salmonella enterica Serovar Typhimurium: Critical Role for Lipopolysaccharide
J. Bacteriol., November 15, 2000; 182(22): 6308 - 6321.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
H. Cai and S. Clarke
A Novel Methyltransferase Catalyzes the Methyl Esterification of trans-Aconitate in Escherichia coli
J. Biol. Chem., May 7, 1999; 274(19): 13470 - 13479.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. K. B. Berlyn
Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map
Microbiol. Mol. Biol. Rev., September 1, 1998; 62(3): 814 - 984.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W.-J. Lin, J. D. Gary, M. C. Yang, S. Clarke, and H. R. Herschman
The Mammalian Immediate-early TIS21 Protein and the Leukemia-associated BTG1 Protein Interact with a Protein-arginine N-Methyltransferase
J. Biol. Chem., June 21, 1996; 271(25): 15034 - 15044.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. D. Gary, W.-J. Lin, M. C. Yang, H. R. Herschman, and S. Clarke
The Predominant Protein-arginine Methyltransferase from Saccharomyces cerevisiae
J. Biol. Chem., May 24, 1996; 271(21): 12585 - 12594.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. D. Lowenson, E. Kim, S. G. Young, and S. Clarke
Limited Accumulation of Damaged Proteins in L-Isoaspartyl (D-Aspartyl) O-Methyltransferase-deficient Mice
J. Biol. Chem., June 1, 2001; 276(23): 20695 - 20702.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. T. Bedford, A. Frankel, M. B. Yaffe, S. Clarke, P. Leder, and S. Richard
Arginine Methylation Inhibits the Binding of Proline-rich Ligands to Src Homology 3, but Not WW, Domains
J. Biol. Chem., May 19, 2000; 275(21): 16030 - 16036.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gary, J. D.
Right arrow Articles by Clarke, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gary, J. D.
Right arrow Articles by Clarke, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement