Planta

, Volume 234, Issue 1, pp 109–122 | Cite as

Structural analysis of K+ dependence in l-asparaginases from Lotus japonicus

  • Alfredo Credali
  • Antonio Díaz-Quintana
  • Margarita García-Calderón
  • Miguel A. De la Rosa
  • Antonio J. Márquez
  • José M. Vega
Original Article

Abstract

The molecular features responsible for the existence in plants of K+-dependent asparaginases have been investigated. For this purpose, two different cDNAs were isolated in Lotus japonicus, encoding for K+-dependent (LjNSE1) or K+-independent (LjNSE2) asparaginases. Recombinant proteins encoded by these cDNAs have been purified and characterized. Both types of asparaginases are composed by two different subunits, α (20 kDa) and β (17 kDa), disposed as (αβ)2 quaternary structure. Major differences were found in the catalytic efficiency of both enzymes, due to the fact that K+ is able to increase by tenfold the enzyme activity and lowers the Km for asparagine specifically in LjNSE1 but not in LjNSE2 isoform. Optimum LjNSE1 activity was found at 5–50 mM K+, with a Km for K+ of 0.25 mM. Na+ and Rb+ can, to some extent, substitute for K+ on the activating effect of LjNSE1 more efficiently than Cs+ and Li+ does. In addition, K+ is able to stabilize LjNSE1 against thermal inactivation. Protein homology modelling and molecular dynamics studies, complemented with site-directed mutagenesis, revealed the key importance of E248, D285 and E286 residues for the catalytic activity and K+ dependence of LjNSE1, as well as the crucial relevance of K+ for the proper orientation of asparagine substrate within the enzyme molecule. On the other hand, LjNSE2 but not LjNSE1 showed β-aspartyl-hydrolase activity (Km = 0.54 mM for β-Asp-His). These results are discussed in terms of the different physiological significance of these isoenzymes in plants.

Keywords

Asparaginases K+-Dependent enzyme activity Lotus Ntn-hydrolases Nitrogen metabolism Homology model 

Abbreviations

BSA

Bovine serum albumin

ESP

Electrostatic surface potential

GOT

Glutamate-oxalacetate transaminase

IPTG

Isopropyl-β-d-thiogalactopyranoside

LjNSE1

Asparaginase 1 from Lotus japonicus

LjNSE2

Asparaginase 2 from Lotus japonicus

M+

Monovalent cations

MD

Molecular dynamics

MDH

Malate dehydrogenase

NPT

Constant pressure and temperature

NVT

Constant volume and temperature

PME

Particle mesh Ewald method

RMSD

Root mean square deviation

RMSF

Root mean square fluctuations

XRD

X-ray diffraction

Supplementary material

425_2011_1393_MOESM1_ESM.ppt (126 kb)
Supplemental Fig. S1 MALDI-MS spectra of purified recombinant LjNSE1 and 2 isoenzymes from L. japonicus. Peaks are identified by numbers corresponding to their molecular weight (PPT 125 kb)
425_2011_1393_MOESM2_ESM.ppt (755 kb)
Supplemental Fig. S2 Dipeptidase activity of LjNSE2 at different β-Asp-His concentrations. The activity was measured as indicated in Material and methods, but using the indicated β-Asp-His concentrations (PPT 755 kb)
425_2011_1393_MOESM3_ESM.ppt (2.6 mb)
Supplemental Fig. S3 Active site pocket in the average structure of MD simulations in presence of K+. Cavity computations were performed as described in methods. The surface is coloured form according to the electrostatic potential: Red negative; white neutral; blue, positive Coulombic potential (PPT 2648 kb)
425_2011_1393_MOESM4_ESM.ppt (881 kb)
Supplemental Fig. S4 Ratio of +K+/-M+ activity corresponding to wild-type and mutants of LjNSE1 isoenzyme. WT (1), D285P(2), E248K(3), E286K(4), E248K-D285P(5), E248K-E286K(6), D285P-E286K(7), and E248K-D285P-E286K(8). Different letters indicate significant difference according to one-way ANOVA (P ≤ 0.01). Error bar means SD (PPT 881 kb)
425_2011_1393_MOESM5_ESM.doc (37 kb)
Supplemental Table S1 Setup of molecular dynamics computations (DOC 37 kb)

References

  1. Aitken A (2005) Identification of protein by MALDI-TOF MS. In: Walker J (ed) Proteomics protocol handbook. Humana Press, Totowa, pp 319–324CrossRefGoogle Scholar
  2. Bendsten JD, Nielsen H, Heijne GH, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795CrossRefGoogle Scholar
  3. Berendsen HJC, Postma JPM, Vangunsteren WF, Dinola A, Haak JR (1984) Molecular-dynamics with coupling to an external bath. J Chem Phys 81:3684–3690CrossRefGoogle Scholar
  4. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242PubMedCrossRefGoogle Scholar
  5. Borek D, Jaskolski M (2001) Sequence analysis of enzymes with asparaginase activity. Acta Biochim Pol 48:893–902PubMedGoogle Scholar
  6. Borek D, Michalska K, Brzecinski K, Kisiel A, Podkowinski J, Bonthron DT, Krowarsch D, Otlewski J, Jaskolski M (2004) Expression, purification and catalytic activity of Lupinus luteus asparagine β-amidohydrolase and its Escherichia coli homolog. Eur J Biochem 271:3215–3226PubMedCrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. Bruneau L, Chapman R, Marsolais F (2006) Co-occurrence of both l-asparaginase subtypes in Arabidopsis: At3g16150 encodes a K+-dependent l-asparaginase. Planta 224:668–679PubMedCrossRefGoogle Scholar
  9. Cañas RA, de la Torre F, Cánovas FM, Cantón FR (2007) Coordination of PsAS1 and PsASPG expression controls timing of re-allocated N utilization in hypocotyls of pine seedlings. Planta 225:1205–1219PubMedCrossRefGoogle Scholar
  10. Case DA, Darden TA, Cheatham TE III, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Pearlman DA, Crowley M, Walker RC, Zhang W, Wang B, Hayyik S, Roitberg A, Seabra G, Wong KF, Paesani F, Wu X, Brozell S, Tsui V, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Beroza P, Mathews DH, Schafmeister C, Ross WS, Kollman PA (2006) AMBER, 9th edn. University of California, San FranciscoGoogle Scholar
  11. Chang KS, Farnden KJF (1981) Purification and properties of asparaginase from Lupinus arboreus and Lupinus angustifolius. Arch Biochem Biophys 208:49–58PubMedCrossRefGoogle Scholar
  12. Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, Liang J (2006) CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acid Res 34 (Web Server issue): W116–W118Google Scholar
  13. Eswar N, Eramian D, Webb B, Shen M-Y, Sali A (2008) Protein structure modelling with MODELLER. Methods Mol Biol 426:145–159PubMedCrossRefGoogle Scholar
  14. Gouet P, Courcelle E, Stuart DI, Métoz F (1999) ESPript: analysis of multiple sequence alignment in PostScript. Bioinformatics 15:305–308PubMedCrossRefGoogle Scholar
  15. Green S, Squire CJ, Nieuwenhuizen NJ, Baker EN, Laing W (2009) Defining the potassium binding region in an apple terpene synthase. J Biol Chem 284:8661–8669PubMedCrossRefGoogle Scholar
  16. Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2:487–496CrossRefGoogle Scholar
  17. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graphics 14:33–38CrossRefGoogle Scholar
  18. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935CrossRefGoogle Scholar
  19. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 12:2577–2637CrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 251:614–616Google Scholar
  21. Lang PT, Brozell SR, Mukherjee S, Pettersen ET, Meng EC, Thomas V, Rizzo RC, Case DA, James TL, Kuntz ID (2009) DOCK 6: combining techniques to model RNA-small molecules complexes. RNA 15:1219–1230PubMedCrossRefGoogle Scholar
  22. Laue TM, Shall BD, Ridgeway TM, Pelletier SL (1992) Computer-aided interpretation of analytical sedimentation data for proteins. In: Harding SE, Rowe AJ, Horton JC (eds) Analytical ultracentrifugation in biochemistry and polymer science. The Royal Society of Chemistry, Cambridge, pp 90–125Google Scholar
  23. Lea PJ, Sodek L, Parry MAJ, Shewry PR, Halford NG (2007) Asparagine in plants. Ann Appl Biol 150:1–26CrossRefGoogle Scholar
  24. Leigh RA, Wyn Jones RG (1984) A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell. New Phytol 97:1–13CrossRefGoogle Scholar
  25. Márquez AJ (2005) Lotus japonicus handbook. Springer, DordrechtGoogle Scholar
  26. Márquez AJ, Betti M, García-Calderón M, Pal’ove-Balang P, Díaz P, Monza J (2005) Nitrate assimilation in Lotus japonicus. J Exp Bot 56:1741–1749PubMedCrossRefGoogle Scholar
  27. Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, LondonGoogle Scholar
  28. Michalska K, Jaskolski M (2006) Structural aspects of l-asparaginases, their friends and relations. Acta Biochim Pol 53:627–640PubMedGoogle Scholar
  29. Michalska K, Brzezinski K, Jaskolski M (2005) Crystal structure of isoaspartylaminopeptidase in complex with l-aspartate. J Biol Chem 280:28484–28491PubMedCrossRefGoogle Scholar
  30. Michalska K, Bujacz G, Jaskolski M (2006) Crystal structure of plant asparaginase. J Mol Biol 360:105–116PubMedCrossRefGoogle Scholar
  31. Michalska K, Hernández-Santoyo A, Jaskolski M (2008) The mechanism of autocatalytic activation of plant-type l-asparaginases. J Biol Chem 283:13388–13397PubMedCrossRefGoogle Scholar
  32. Möllering H (1985) L-aspartate and l-asparagine. In: Bergmeyer HU, Bergmeyer J, Grass IM (eds) Methods of enzymatic analysis, vol VIII, 3rd edn. VCH Verlagsgessellschaft, Weinheim, pp 350–357Google Scholar
  33. Nielsen H, Engelbrech J, Brunak S, Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6PubMedCrossRefGoogle Scholar
  34. Oza VP, Trivedi SD, Parmar PP, Subramanian RB (2009) Withania somnifera (ashwagandha): a novel source of l-asparaginase. J Integrative Plant Biol 51:201–206CrossRefGoogle Scholar
  35. Oza VP, Parmar PP, Kumar S, Subramanian RB (2010) Anticancer properties of highly purified l-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia. Appl Biochem Biotechnol 160:1833–1840PubMedCrossRefGoogle Scholar
  36. Page MJ, Di Cera E (2006) Role of Na+ and K+ in enzyme function. Physiol Rev 86:1049–1092PubMedCrossRefGoogle Scholar
  37. Pettersen EFG, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612PubMedCrossRefGoogle Scholar
  38. Philo JS (1997) An improved function for fitting sedimentation velocity data for low molecular-weight solutes. Biophys J 72:435–444PubMedCrossRefGoogle Scholar
  39. Ryckaert JP, Ciccoti G, Berendsen HJC (1977) Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys 23:327–341CrossRefGoogle Scholar
  40. Sodek L, Lea PJ, Miflin BJ (1980) Distribution and properties of a potassium-dependent asparaginase isolated from developing seeds of Pisum sativum and other plants. Plant Physiol 65:22–26PubMedCrossRefGoogle Scholar
  41. Stacey G, Libault M, Brechenmacher L, Wan J, May GD (2006) Genetics and functional genomics of legume nodulation. Curr Opin Plant Biol 9:110–121PubMedCrossRefGoogle Scholar
  42. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  43. Udvardi MK, Tabata S, Parniske M, Stougaard J (2005) Lotus japonicus: legume research in the fast line. Trends Plant Sci 10:222–228PubMedCrossRefGoogle Scholar
  44. Verma N, Kumar K, Kaur G, Anand S (2007) l-Asparaginase: a promising chemotherapeutic agent. Crit Rev Biotechnol 27:45–62PubMedCrossRefGoogle Scholar
  45. Wang JM, Cieplak P, Kollman PA (2000) How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comput Chem 21:1049–1074CrossRefGoogle Scholar
  46. Waterhouse RN, Smyth AJ, Massonneau A, Prosser IM, Clarkson DT (1996) Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: dynamics of asparagine synthesis in N-sufficient conditions. Plant Mol Biol 30:883–897PubMedCrossRefGoogle Scholar
  47. Williams NH (1998) Phosphate diesterases and triesterases. In: Sinnot M (ed) Comprehensive biological catalysis, vol 1. Reaction of electrophilic carbon, phosphorus and sulfur. Academic Press, London, pp 543–557Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Alfredo Credali
    • 1
  • Antonio Díaz-Quintana
    • 2
  • Margarita García-Calderón
    • 1
  • Miguel A. De la Rosa
    • 2
  • Antonio J. Márquez
    • 1
  • José M. Vega
    • 1
  1. 1.Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de QuímicaUniversidad de SevillaSevilleSpain
  2. 2.Instituto de Bioquímica Vegetal y FotosíntesisCentro de Investigaciones Científicas, Isla de la CartujaSevilleSpain

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