title: Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.
abstract: 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.
authors.affiliation: From the Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612 and the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607.
authors.forename: Hien Anh
authors.initials: HA
authors.lastname: Nguyen
authors.affiliation: From the Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612 and the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607.
authors.forename: Ying
authors.initials: Y
authors.lastname: Su
authors.affiliation: From the Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612 and the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 Lavie@uic.edu.
authors.forename: Arnon
authors.initials: A
authors.lastname: Lavie
mesh_terms: D019943:Amino Acid Substitution; D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D045744:Cell Line, Tumor; D016974:Dickeya chrysanthemi / Q000201:enzymology* / Q000235:genetics; D005972:Glutaminase; D006801:Humans; D020125:Mutation, Missense
chemical_list: D001426:Bacterial Proteins; D001215:Asparaginase; D005972:Glutaminase
keywords: cancer therapy; enzyme kinetics; enzyme mutation; leukemia; structural biology; structure-function; substrate specificity