Previous Article | Next Article 
Journal of Bacteriology, February 2001, p. 1489-1490, Vol. 183, No. 4
Division of Integrated Life Science, Graduate
School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto 606-8502, Japan
Received Recieved 30 October 2000/Accepted 21 November 2000
Aminopeptidases A, B, and N and dipeptidase D, with broad substrate
specificity, are the four cysteinylglycinases of Escherichia coli K-12, and there is no peptidase specific for the cleavage of cysteinylglycine.
Glutathione is a tripeptide with the
structure L- L-Cysteinylglycine and L-leucylglycine were
purchased from Sigma Chemical Co. All strains used were E. coli K-12 derivatives and are listed in Table
1.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.4.1489-1490.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Aminopeptidases A, B, and N and Dipeptidase D Are
the Four Cysteinylglycinases of Escherichia coli
K-12
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
-glutamyl-L-cysteinylglycine.
Escherichia coli K-12 synthesizes glutathione and, during
the exponential and early stationary phases, excretes into the medium
some glutathione (9, 16), which is subsequently utilized
during the stationary phase (16).
-Glutamyltranspeptidase existing in the periplasm (14)
cleaves the -glutamyl linkage of glutathione to generate glutamate
and cysteinylglycine, and cysteinylglycine is taken up into the
cytoplasm and utilized as both cysteine and glycine sources
(12). In a previous paper, we proposed that this is the
cysteine salvage pathway in E. coli K-12 (13).
Thus, the next step was to identify which peptidase of E. coli K-12 is responsible for the cleavage of the peptide bond of
cysteinylglycine between cysteine and glycine. Since McCorquodale's description of cysteinylglycinase activity in E. coli B
(3), there have been no reports on cysteinylglycinase of
E. coli. Miller and his coworkers performed an extensive
study on peptidases of E. coli K-12 and Salmonella
enterica serovar Typhimurium using peptidase-deficient strains and
elucidated their physiological roles (reviews in references 4 and
5). They reported that one of the physiological roles of
cytoplasmic dipeptidases and aminopeptidases is the hydrolysis of
peptides supplied exogenously, which allows the peptides to be used as
amino acid sources. However, although they investigated the substrate
specificity of peptidases, they did not investigate whether these
peptidases are able to cleave the peptide bond of cysteinylglycine or
if there is another cysteinylglycinase different from the peptidases
they described (7). In this study, the peptidases of
E. coli K-12 responsible for the cleavage of
cysteinylglycine were identified.
(pro-lac) deletes the
pepD gene (7). All pep mutants were
grown in Luria-Bertani broth (8) supplemented with 0.05 mM
thymine and 0.03 mM thiamine at 37°C. As a minimal medium, M9 glucose
medium (8) supplemented with 0.05 mM leucine, 0.3 mM
methionine, 0.3 mM proline, 0.05 mM thymine, and 0.03 mM thiamine was
used. When necessary, antibiotics and peptides were added. KES and SH
strains were constructed by P1 vir-mediated transduction and
Hfr mating (Table 1) as described previously (16).
E. coli K-12 lacks valine-resistant acetohydroxy acid synthase and cannot grow on a minimal medium containing valine unless
isoleucine is added (valine sensitivity of E. coli K-12) (17). A pepABDN mutant, such as strain CM86, is
valylvaline resistant because only peptidases A, B, D, and N of
E. coli K-12 can cleave the peptide bond of this dipeptide
to liberate valine (7); in addition, reversion of
any one of these peptidase genes makes the strain sensitive to
valylvaline (at 0.25 mM). Therefore, when the
pepA+, pepB+,
pepD+, or pepN+ allele
was introduced into a pepABDN strain by transduction or Hfr
mating, tetracycline-resistant transductants and transconjugants were
screened for valylvaline resistance and valylvaline-sensitive transductants and transconjugants were stored as Pep+
strains. Cell extracts of these Pep+ strains were subjected
to native polyacrylamide gel electrophoresis (15),
followed by peptidase activity staining using
L-leucylglycine as a substrate (6). The
peptidase bands formed were compared with those of the control strain
to confirm that the Pep+ phenotype was derived from the
desired pep+ gene introduced into the strain.
The assay solution for cysteinylglycinase activity was comprised of 0.5 mM cysteinylglycine, 50 mM Tris-HCl (pH 7.5), and 1 mM
MnSO4, in a final volume of 0.1 ml. The reaction was
carried out at 37°C and was terminated by the addition of 0.9 ml of
0.5 M potassium citrate buffer (pH 2.2). The amount of glycine released
was measured with a high-performance liquid chromatograph equipped with
a Shim-pack Amino-Na column and a fluorescence detector (model LC-9A;
Shimadzu, Kyoto, Japan) with o-phthalaldehyde as the
detection reagent. One unit of enzyme was defined as the amount of
enzyme that released 1 µmol of glycine per min. Protein
concentrations were measured by the method of Lowry et al.
(2), with bovine serum albumin as a standard.
TABLE 1.
E. coli K-12 strains used in this study
Aminopeptidases A, B, and N and dipeptidase D are known as peptidases
with broad substrate specificity (7). Strain CM86, which
has defects in all of these peptidases, showed no detectable cysteinylglycinase activity (Table 2).
Strains that recovered one of these four peptidases were constructed,
and their cysteinylglycinase activities were measured. All four strains
recovered cysteinylglycinase activity (Table 2).
|
Cysteine auxotrophy was introduced into these strains, and utilization
of cysteinylglycine as a cysteine source was tested (Table
3). Strain SH1420 could not grow on the
minimal medium supplemented with cysteinylglycine as a cysteine source,
while SH1429, SH1423, SH1424, and SH1426, which recovered
peptidases A, B, N, and D, respectively, grew on the same plate.
|
These results indicate that there is no peptidase specific for the cleavage of cysteinylglycine, but that any one of aminopeptidase A, B, or N or dipeptidase D is sufficient for E. coli to utilize cysteinylglycine as a cysteine source.
Using S. enterica serovar Typhimurium strain TA100, Glatt et
al. found that glutathione in the presence of rat kidney homogenate was
Ames test positive (1). Stark et al. showed that
cysteinylglycine generated through the cleavage of glutathione by
-glutamyltranspeptidase is subjected to auto-oxidation, with the
production of free radicals that leads to hydrogen peroxide, the
ultimate mutagen (11). Since strain CM86 was found to have
no detectable cysteinylglycinase activity, the question of whether CM86
is more mutagenic than the control strain arose. The frequency of
appearance of streptomycin-resistant mutants on the medium containing
cysteinylglycine did not differ between strain CM86 and the
control strain (data not shown). Although these pep
mutations in the S. enterica serovar Typhimurium TA100 and
TA102 backgrounds should be investigated, in our strain background, a
deficiency of cysteinylglycinase had no effect on the mutagenicity of cysteinylglycine.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by Grants-in-Aid for Scientific Research no. 10660083 to H.S. and no. 10306007 to H.K. from the Ministry of Education, Science, and Culture of Japan.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81-75-753-6278. Fax: 81-75-753-6275. E-mail: hideyuki{at}lif.kyoto-u.ac.jp.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Glatt, H.,
C. M. Protic-Sablji, and F. Oesch.
1983.
Mutagenicity of glutathione and cysteine in the Ames test.
Science
220:961-963 |
| 2. |
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275 |
| 3. |
McCorquodale, D. J.
1963.
Some properties of a ribosomal cysteinylglycinase of Escherichia coli B.
J. Biol. Chem.
238:3914-3920 |
| 4. |
Miller, C. G.
1985.
Genetics and physiological roles of Salmonella typhimurium peptidases, p. 346-349.
In
L. Leive (ed.), Microbiology 1985. American Society for Microbiology, Washington, D.C.
|
| 5. | Miller, C. G. 1996. Protein degradation and proteolytic modification, p. 938-954. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Rilley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 1. American Society for Microbiology, Washington, D.C. |
| 6. |
Miller, C. G., and K. Mackinnon.
1974.
Peptidase mutants of Salmonella typhimurium.
J. Bacteriol.
120:355-363 |
| 7. |
Miller, C. G., and G. Schwartz.
1978.
Peptidase-deficient mutants of Escherichia coli.
J. Bacteriol.
135:603-611 |
| 8. | Miller, J. H. 1992. A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 9. |
Owens, R. A., and P. E. Hartman.
1986.
Export of glutathione by some widely used Salmonella typhimurium and Escherichia coli strains.
J. Bacteriol.
168:109-114 |
| 10. |
Singer, M.,
T. A. Baker,
G. Schnitzler,
S. M. Deischel,
M. Goel,
W. Dove,
K. J. Jaacks,
A. D. Grossman,
J. W. Erickson, and C. A. Gross.
1989.
A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.
Microbiol. Rev.
53:1-24 |
| 11. |
Stark, A. A.,
E. Zeiger, and D. A. Pagano.
1988.
Glutathione mutagenesis in Salmonella typhimurium is a -glutamyltranspeptidase-enhanced process involving active oxygen species.
Carcinogenesis
9:771-777 |
| 12. |
Suzuki, H.,
W. Hashimoto, and H. Kumagai.
1993.
Escherichia coli K-12 can utilize an exogenous -glutamyl peptide as an amino acid source, for which -glutamyltranspeptidase is essential.
J. Bacteriol.
175:6038-6040 |
| 13. | Suzuki, H., W. Hashimoto, and H. Kumagai. 1999. Glutathione metabolism in Escherichia coli. J. Mol. Catal. B 6:175-184[CrossRef]. |
| 14. |
Suzuki, H.,
H. Kumagai, and T. Tochikura.
1986.
-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization.
J. Bacteriol.
168:1332-1335 |
| 15. |
Suzuki, H.,
H. Kumagai, and T. Tochikura.
1986.
-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties.
J. Bacteriol.
168:1325-1331 |
| 16. |
Suzuki, H.,
H. Kumagai, and T. Tochikura.
1987.
Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking -glutamyltranspeptidase.
J. Bacteriol.
169:3926-3931 |
| 17. | Umbarger, H. E. 1983. The biosynthesis of isoleucine and valine and its regulation, p. 245-266. In K. M. Herrmann, and R. L. Somerville (ed.), Amino acids biosynthesis and genetic regulation. Addison-Wesley Publishing, Reading, Mass. |
Journal of Bacteriology, February 2001, p. 1489-1490, Vol. 183, No. 4
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.4.1489-1490.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Kaur, H., Kumar, C., Junot, C., Toledano, M. B., Bachhawat, A. K.
(2009). Dug1p Is a Cys-Gly Peptidase of the {gamma}-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases. J. Biol. Chem.
284: 14493-14502
[Abstract] [Full Text] -
Chu, L., Lai, Y., Xu, X., Eddy, S., Yang, S., Song, L., Kolodrubetz, D.
(2008). A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism. J. Biol. Chem.
283: 19351-19358
[Abstract] [Full Text] -
Tabata, K., Hashimoto, S.-i.
(2007). Fermentative Production of L-Alanyl-L-Glutamine by a Metabolically Engineered Escherichia coli Strain Expressing L-Amino Acid {alpha}-Ligase. Appl. Environ. Microbiol.
73: 6378-6385
[Abstract] [Full Text] -
Suzuki, H., Koyanagi, T., Izuka, S., Onishi, A., Kumagai, H.
(2005). The yliA, -B, -C, and -D Genes of Escherichia coli K-12 Encode a Novel Glutathione Importer with an ATP-Binding Cassette. J. Bacteriol.
187: 5861-5867
[Abstract] [Full Text] -
Takahashi, H., Hirose, K., Watanabe, H.
(2004). Necessity of Meningococcal {gamma}-Glutamyl Aminopeptidase for Neisseria meningitidis Growth in Rat Cerebrospinal Fluid (CSF) and CSF-Like Medium. J. Bacteriol.
186: 244-247
[Abstract] [Full Text] -
Chandu, D., Nandi, D.
(2003). PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress. Microbiology
149: 3437-3447
[Abstract] [Full Text] -
Chandu, D., Kumar, A., Nandi, D.
(2003). PepN, the Major Suc-LLVY-AMC-hydrolyzing Enzyme in Escherichia coli, Displays Functional Similarity with Downstream Processing Enzymes in Archaea and Eukarya. IMPLICATIONS IN CYTOSOLIC PROTEIN DEGRADATION. J. Biol. Chem.
278: 5548-5556
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»