, 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


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.


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



Bovine serum albumin


Electrostatic surface potential


Glutamate-oxalacetate transaminase




Asparaginase 1 from Lotus japonicus


Asparaginase 2 from Lotus japonicus


Monovalent cations


Molecular dynamics


Malate dehydrogenase


Constant pressure and temperature


Constant volume and temperature


Particle mesh Ewald method


Root mean square deviation


Root mean square fluctuations


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)


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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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