Development of new candidate oncological therapeutic enzymes: A comparative structural and kinetic study of recombinant L-asparaginases

L-asparaginases (L-ASNase, EC 3.5.1.1) are amidohydrolases catalysing the conversion of L-Asn to L-Asp and ammonia and to a lower extent L-Gln to L-Glu (L-glutaminase activity, L-GLNase). This enzyme is exploited as an anti-neoplastic agent to treat acute lymphoblastic leukaemia. Currently, Escherichia coli and Erwinia chrysanthemi L-ASNases are used as oncological drugs. However, harmful side-effects and hypersensitivity reactions are the main limitations of these therapeutics. A link has been proposed between L-GLNase activity and harmful side effects. Therefore, finding of new L-ASNases with low glutaminase activity, thus potentially with lower side effect risk, is important for medical applications. A detailed biochemical characterization of a wide range of L-ASNases linked with in silico approaches could contribute for discovering better oncologic ASNase drug candidates. In this study, ten bacterial and yeast L-ASNase genes belonging to type I and II class of L-ASNase families, were synthesized and cloned into T7 bacterial expression vector for production in E. coli BL21 cells. The optimum pH and temperature of the expressed L-ASNases were at pH 7.0-9.0 and 35°C-50°C range, respectively. While the optimum temperature and pH of the ten L-ASNases were similar as those previously reported for type I and II L-ASNases, none of them displayed detectable glutaminase activity. Structural comparisons of these ten ASNases with quite much differing kinetic properties showed that the residues with a catalytic role are conserved and some differences at position 59 close to the substrate may affect the kinetic parameters. The type I L-ASNase from Lachancea thermotolerans yeast ( LtASNase) exhibited the highest specific activity of 313.82 U/mg and catalytic efficiency for L-Asn. Therefore, LtASNase is a promising enzyme for further investigation in pharmaceutical applications.