Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

J Biol Chem. 2016 Aug 19;291(34):17664-76. doi: 10.1074/jbc.M116.728485. Epub 2016 Jun 27.


Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects.

Keywords: cancer therapy; enzyme kinetics; enzyme mutation; leukemia; structural biology; structure-function; substrate specificity.

MeSH terms

  • Amino Acid Substitution
  • Asparaginase / chemistry*
  • Asparaginase / genetics
  • Asparaginase / pharmacology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / pharmacology
  • Cell Line, Tumor
  • Dickeya chrysanthemi / enzymology*
  • Dickeya chrysanthemi / genetics
  • Glutaminase*
  • Humans
  • Mutation, Missense*


  • Bacterial Proteins
  • Asparaginase
  • Glutaminase

Associated data

  • PDB/1O7J
  • PDB/5F52
  • PDB/5HW0
  • PDB/5I3Z
  • PDB/5I48
  • PDB/5I4B