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. 2002 May 15;364(Pt 1):129–136. doi: 10.1042/bj3640129

Isoaspartyl dipeptidase activity of plant-type asparaginases.

Mahdi Hejazi 1, Kirill Piotukh 1, Jens Mattow 1, Rainer Deutzmann 1, Rudolf Volkmer-Engert 1, Wolfgang Lockau 1
PMCID: PMC1222554  PMID: 11988085

Abstract

Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp. PCC (Pasteur culture collection) 6803 and Anabaena sp. PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E. coli with either an N-terminal or a C-terminal His tag, and purified. Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly-Thr bond within the sequence GTI/VG. The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides. As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation. The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.

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Selected References

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  1. Allen M. B., Arnon D. I. Studies on Nitrogen-Fixing Blue-Green Algae. I. Growth and Nitrogen Fixation by Anabaena Cylindrica Lemm. Plant Physiol. 1955 Jul;30(4):366–372. doi: 10.1104/pp.30.4.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aswad D. W., Paranandi M. V., Schurter B. T. Isoaspartate in peptides and proteins: formation, significance, and analysis. J Pharm Biomed Anal. 2000 Jan;21(6):1129–1136. doi: 10.1016/s0731-7085(99)00230-7. [DOI] [PubMed] [Google Scholar]
  4. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  5. Brannigan J. A., Dodson G., Duggleby H. J., Moody P. C., Smith J. L., Tomchick D. R., Murzin A. G. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature. 1995 Nov 23;378(6555):416–419. doi: 10.1038/378416a0. [DOI] [PubMed] [Google Scholar]
  6. Chang K. S., Farnden K. J. Purification and properties of asparaginase from Lupinus arboreus and Lupinus angustifolius. Arch Biochem Biophys. 1981 Apr 15;208(1):49–58. doi: 10.1016/0003-9861(81)90122-3. [DOI] [PubMed] [Google Scholar]
  7. Dorer F. E., Haley E. E., Buchanan D. L. The hydrolysis of beta-aspartyl peptides by rat tissue. Arch Biochem Biophys. 1968 Sep 20;127(1):490–495. doi: 10.1016/0003-9861(68)90253-1. [DOI] [PubMed] [Google Scholar]
  8. Gary J. D., Clarke S. Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli. J Biol Chem. 1995 Feb 24;270(8):4076–4087. doi: 10.1074/jbc.270.8.4076. [DOI] [PubMed] [Google Scholar]
  9. Guan C., Liu Y., Shao Y., Cui T., Liao W., Ewel A., Whitaker R., Paulus H. Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase. J Biol Chem. 1998 Apr 17;273(16):9695–9702. doi: 10.1074/jbc.273.16.9695. [DOI] [PubMed] [Google Scholar]
  10. Guo H. C., Xu Q., Buckley D., Guan C. Crystal structures of Flavobacterium glycosylasparaginase. An N-terminal nucleophile hydrolase activated by intramolecular proteolysis. J Biol Chem. 1998 Aug 7;273(32):20205–20212. doi: 10.1074/jbc.273.32.20205. [DOI] [PubMed] [Google Scholar]
  11. Jungblut P. R., Schaible U. E., Mollenkopf H. J., Zimny-Arndt U., Raupach B., Mattow J., Halada P., Lamer S., Hagens K., Kaufmann S. H. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol Microbiol. 1999 Sep;33(6):1103–1117. doi: 10.1046/j.1365-2958.1999.01549.x. [DOI] [PubMed] [Google Scholar]
  12. Kaneko T., Sato S., Kotani H., Tanaka A., Asamizu E., Nakamura Y., Miyajima N., Hirosawa M., Sugiura M., Sasamoto S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 1996 Jun 30;3(3):109–136. doi: 10.1093/dnares/3.3.109. [DOI] [PubMed] [Google Scholar]
  13. Larsen R. A., Knox T. M., Miller C. G. Aspartic peptide hydrolases in Salmonella enterica serovar typhimurium. J Bacteriol. 2001 May;183(10):3089–3097. doi: 10.1128/JB.183.10.3089-3097.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lough T. J., Reddington B. D., Grant M. R., Hill D. F., Reynolds P. H., Farnden K. J. The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus). Plant Mol Biol. 1992 Jun;19(3):391–399. doi: 10.1007/BF00023386. [DOI] [PubMed] [Google Scholar]
  15. Noronkoski T., Stoineva I. B., Ivanov I. P., Petkov D. D., Mononen I. Glycosylasparaginase-catalyzed synthesis and hydrolysis of beta-aspartyl peptides. J Biol Chem. 1998 Oct 9;273(41):26295–26297. doi: 10.1074/jbc.273.41.26295. [DOI] [PubMed] [Google Scholar]
  16. Noronkoski T., Stoineva I. B., Petkov D. D., Mononen I. Recombinant human glycosylasparaginase catalyzes hydrolysis of L-asparagine. FEBS Lett. 1997 Jul 21;412(1):149–152. doi: 10.1016/s0014-5793(97)00761-8. [DOI] [PubMed] [Google Scholar]
  17. Oinonen C., Rouvinen J. Structural comparison of Ntn-hydrolases. Protein Sci. 2000 Dec;9(12):2329–2337. doi: 10.1110/ps.9.12.2329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oinonen C., Tikkanen R., Rouvinen J., Peltonen L. Three-dimensional structure of human lysosomal aspartylglucosaminidase. Nat Struct Biol. 1995 Dec;2(12):1102–1108. doi: 10.1038/nsb1295-1102. [DOI] [PubMed] [Google Scholar]
  19. Richter R., Hejazi M., Kraft R., Ziegler K., Lockau W. Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartic acid (cyanophycin): molecular cloning of the gene of Synechocystis sp. PCC 6803, expression in Escherichia coli, and biochemical characterization of the purified enzyme. Eur J Biochem. 1999 Jul;263(1):163–169. doi: 10.1046/j.1432-1327.1999.00479.x. [DOI] [PubMed] [Google Scholar]
  20. Rippka R. Isolation and purification of cyanobacteria. Methods Enzymol. 1988;167:3–27. doi: 10.1016/0076-6879(88)67004-2. [DOI] [PubMed] [Google Scholar]
  21. Sørensen S. B., Sørensen T. L., Breddam K. Fragmentation of proteins by S. aureus strain V8 protease. Ammonium bicarbonate strongly inhibits the enzyme but does not improve the selectivity for glutamic acid. FEBS Lett. 1991 Dec 9;294(3):195–197. doi: 10.1016/0014-5793(91)80667-r. [DOI] [PubMed] [Google Scholar]
  22. Tarentino A. L., Quinones G., Hauer C. R., Changchien L. M., Plummer T. H., Jr Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits. Arch Biochem Biophys. 1995 Jan 10;316(1):399–406. doi: 10.1006/abbi.1995.1053. [DOI] [PubMed] [Google Scholar]
  23. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Xu Q., Buckley D., Guan C., Guo H. C. Structural insights into the mechanism of intramolecular proteolysis. Cell. 1999 Sep 3;98(5):651–661. doi: 10.1016/s0092-8674(00)80052-5. [DOI] [PubMed] [Google Scholar]
  25. Ziegler K., Diener A., Herpin C., Richter R., Deutzmann R., Lockau W. Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). Eur J Biochem. 1998 May 15;254(1):154–159. doi: 10.1046/j.1432-1327.1998.2540154.x. [DOI] [PubMed] [Google Scholar]
  26. Zuther E., Schubert H., Hagemann M. Mutation of a gene encoding a putative glycoprotease leads to reduced salt tolerance, altered pigmentation, and cyanophycin accumulation in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol. 1998 Apr;180(7):1715–1722. doi: 10.1128/jb.180.7.1715-1722.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

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