title: Recombinant deamidated mutants of Erwinia chrysanthemi L-asparaginase have similar or increased activity compared to wild-type enzyme.
abstract: The enzyme Erwinia chrysanthemi L-asparaginase (ErA) is an important biopharmaceutical product used in the treatment of acute lymphoblastic leukaemia. Like all proteins, certain asparagine (Asn) residues of ErA are susceptible to deamidation to aspartic acid (Asp), which may be a concern with respect to enzyme activity and potentially to pharmaceutical efficacy. Recombinant ErA mutants containing Asn to Asp changes were expressed, purified and characterised. Two mutants with single deamidation sites (N41D and N281D) were found to have approximately the same specific activity (1,062 and 924 U/mg, respectively) as the wild-type (908 U/mg). However, a double mutant (N41D N281D) had an increased specific activity (1261 U/mg). The N41D mutation conferred a slight increase in the catalytic constant (k cat 657 s(-1)) when compared to the WT (k cat 565 s(-1)), which was further increased in the double mutant, with a k cat of 798 s(-1). Structural analyses showed that the slight changes caused by point mutation of Asn41 to Asp may have reduced the number of hydrogen bonds in this α-helical part of the protein structure, resulting in subtle changes in enzyme turnover, both structurally and catalytically. The increased α-helical content observed with the N41D mutation by circular dichroism spectroscopy correlates with the difference in k cat, but not K m. The N281D mutation resulted in a lower glutaminase activity compared with WT and the N41D mutant, however the N281D mutation also imparted less stability to the enzyme at elevated temperatures. Taken as a whole, these data suggest that ErA deamidation at the Asn41 and Asn281 sites does not affect enzyme activity and should not be a concern during processing, storage or clinical use. The production of recombinant deamidated variants has proven an effective and powerful means of studying the effect of these changes and may be a useful strategy for other biopharmaceutical products.
authors.affiliation: Microbiology Services, Development & Production, Public Health England, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK, dave.gervais@phe.gov.uk.
authors.forename: David
authors.initials: D
authors.lastname: Gervais
authors.forename: Nicholas
authors.initials: N
authors.lastname: Foote
mesh_terms: D000577:Amides; D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006860:Hydrogen Bonding / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000577:Amides; D050505:Mutant Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D001215:Asparaginase