Rhodospirillum rubrum L-Asparaginase Mutant Forms with Improved Conformation Stability, Biocatalytic Properties and Enhanced Cytostatic Activity

Structural features, underlying the mismatch between catalytic and cytostatic proper-ties in L-asparaginase from Rhodospirillum rubrum (RrA) and three of its mutants were investigated. The effect of point substitutions in the area of inter-subunit contacts of the enzyme (Mut1: A64V, E67K and Mut2: R118H, G120R) as well as in the region, which is probably involved in a contact area upon interaction of the enzyme with the cancer cells (Mut3: E149R, V150P, F151T) has been studied. RrA Mut1 and Mut 2 additionally con-tained an N-terminal 17-amino acid capsid peptide derived from the bacteriophage T7 (MASMTGGQQMGRGSSRQ), which could presumably impact to the conformational stability of the enzymes. Fourier-transform infrared (FTIR) spectroscopy revealed minor alterations in the secondary structure of the RrA mutants compared to the wild-type. Circular dichroism (CD) spectroscopy applied to the kinetic parameters analysis of Asn hydrolysis, showed that native RrA displayed a Vmax of 30 U/mg and a KM of 4.5 ± 0.5 mM. RrA Mut3 exhibited a substantially increased Vmax of 57 U/mg, whereas other mutants exhibited less pronounced changes. Thermo-denaturation studies, allowed to determine the phase transition parameters of the RrA variants in comparison with commercial EcA. The mutants RrA-Mut1 and RrA-Mut2 exhibit the most favorable phase transition parameters, with melting temperatures (Tm) of 60.3 °C and 59.4 °C, respectively, ex-ceeding that of the wild-type RrA (54.6 °C) and RrA-Mut3 (52 °C). EcA demonstrates slightly superior thermal stability, with a Tm of 62 °C. The mutations show significant effect on the protein stability to trypsinolysis. So, Mut3 showed significantly higher resistance (45% activity remaining after 30 min of trypsin exposure) compared to the native RrA retained 20% activity. EcA preparations exhibited lower stability to trypsi-nolysis (losing over 90% activity within 15 min). The cytostatic effects were evaluated using MTT assays against K562 (leukemic) and A549 (lung carcinoma) cell lines. MTT assays with K562 cells revealed that RrA Mut3 (IC50 of 10 U/mL) and RrA Mut2 (IC50 of 11.5 U/mL) exhibited superior anti-proliferative activity compared to native enzymes RrA (IC50 of 15 U/mL) and EcA-Veropharm (24 U/mL). RrA-Mut3 shows the most significant improvement in cytostatic activity. The results obtained indicate that the sub-stitutions in Mut3: E149R, V150P, F151T not only stabilize the conformation of the protein and increase resistance to trypsinolysis, but more importantly, this region is apparently involved in interaction with cancer cells.