The E. coli L-asparaginase V27T mutant: structural and functional characterization and comparison with theoretical predictions

Abstract

Bacterial L-asparaginases have been used for over 40 years as anticancer drugs. Ardalan et al. (Medical Hypotheses 112, 7-17, 2018) proposed that the V27T mutant of Escherichia coli type II L-asparaginase, EcAII(V27T), should display altered biophysical and catalytic properties compared to the wild-type enzyme, EcAII(wt), rendering it more favourable as a pharmaceutical. They postulated that EcAII(V27T) would exhibit reduced glutaminolytic activity and be more stable compared to EcAII(wt). Their postulates, however, were purely theoretical. Here, we characterized experimentally selected properties of EcAII(V27T). We found asparaginolytic activity of this mutant unchanged, whereas its glutaminolytic activity was fourfold lower compared with EcAII(wt). We did not observe significant differences in stabilities of EcAII(wt) and EcAII(V27T). Crystal structures of the complexes with L-Asp and L-Glu showed considerable differences in binding modes of both substrates.

Keywords: X-ray crystallography; asparaginolytic activity; bacterial L-asparaginases; biological assembly; glutaminolytic activity; single-site mutation.

Fig. 1.

Topological features of EcAII(V27T). (A) a cartoon representation of the EcAII(V27T)/L-Asp complex, formed by association of two tight dimers, coloured orange (upper section) and green (at the bottom) respectively. (B) Superimposed structures of EcAII(V27T)/L-Asp (yellow/orange) and EcAII(V27T)/L-Glu (magenta/cyan). N termini (coloured blue), active site flexible loop (ASFL, coloured green), the region Val30-Ala38 (coloured red) and the active site pocket (dashed circle) are indicated.

Fig. 2.

Structure of EcAII(V27T) in complex with L-Asp. (A) Superimposed structures of EcAII(V27T) (protomer a orange and protomer B cyan) and EcAII(wt) (PDB ID 6pac; protomer a red; protomer B green) in complex with L-Asp. The amino acids involved in the stabilizing the ASFL are shown in the inset. (B) The L-Asp molecule bound to the EcAII(V27T) active site, covered by the mFo-DFc omit map contoured at 3.0r level. Important interactions (mostly H-bonds) between the L-Asp and active site residues are indicated by black dashed lines with corresponding distances. *Residues contributed by another protomer from the same tight dimer.

Fig. 3.

Interactions between L-Glu and the active site of EcAII(V27T). (A) Active site of EcAII(V27T) with bound L-Glu molecule. The ligand is shown together with associated mFo-DFc omit map, contoured at 3.0 σ level. (B) L-Glu (purple bonds) interacts with the protein via an extensive network of hydrogen bonds (dashed, green lines; distances are given in A). Hydrophobic interactions are indicated by ‘eyelashes’. Letters in parentheses refer to chain IDs.