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Identification of Functional Regions in the Rhodospirillum rubrum l-Asparaginase by Site-Directed Mutagenesis

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Abstract

Site-directed mutagenesis of Rhodospirillum rubrum l-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70–80 %, their specific activity at pH 7.4 and 37 °C varied of 140–210 U/mg. l-Glutaminase activity did not exceed 0.01 % of l-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3–9.5 and 53–58 °C. Km and Vmax values for l-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.

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Acknowledgments

We express our sincere gratitude to Dr. Mikhail A. Eldarov (Centre “Bioengineering” RAS, Moscow, Russia) for his participation in the discussion, constructive suggestions, useful critique of this research work as well as for help with translation of this article. Dr. Vasiliy N. Lazarev’s (Scientific Research Institute of Physical–Chemical Medicine, Moscow, Russia) valuable support and assistance in DNA sequencing and Prof. Ekaterina Kolesanova’s (Orekhovich Institute of Biomedical Chemistry, Moscow, Russia) help with protein oligomerisation studies are greatly acknowledged.

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  1. Institute of Biomedical Chemistry, ul. Pogodinskaya 10, Moscow, 119121, Russia

    M. V. Pokrovskaya, S. S. Aleksandrova, V. S. Pokrovsky, A. V. Veselovsky, D. V. Grishin, O. Yu. Abakumova, O. V. Podobed, A. A. Mishin, D. D. Zhdanov & N. N. Sokolov

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  1. M. V. Pokrovskaya
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  2. S. S. Aleksandrova
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  3. V. S. Pokrovsky
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  6. O. Yu. Abakumova
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Pokrovskaya, M.V., Aleksandrova, S.S., Pokrovsky, V.S. et al. Identification of Functional Regions in the Rhodospirillum rubrum l-Asparaginase by Site-Directed Mutagenesis. Mol Biotechnol 57, 251–264 (2015). https://doi.org/10.1007/s12033-014-9819-0

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