Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV
The ReAV enzyme from <i>Rhizobium etli</i>, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each sub...
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2025-06-01
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| author | Kinga Pokrywka Marta Grzechowiak Joanna Sliwiak Paulina Worsztynowicz Joanna I. Loch Milosz Ruszkowski Miroslaw Gilski Mariusz Jaskolski |
| author_facet | Kinga Pokrywka Marta Grzechowiak Joanna Sliwiak Paulina Worsztynowicz Joanna I. Loch Milosz Ruszkowski Miroslaw Gilski Mariusz Jaskolski |
| author_sort | Kinga Pokrywka |
| collection | DOAJ |
| description | The ReAV enzyme from <i>Rhizobium etli</i>, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism. |
| format | Article |
| id | doaj-art-a873a0ef5c2c4c868d8a225ffae8dc32 |
| institution | DOAJ |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
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| series | Biomolecules |
| spelling | doaj-art-a873a0ef5c2c4c868d8a225ffae8dc322025-08-20T02:45:45ZengMDPI AGBiomolecules2218-273X2025-06-0115794410.3390/biom15070944Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAVKinga Pokrywka0Marta Grzechowiak1Joanna Sliwiak2Paulina Worsztynowicz3Joanna I. Loch4Milosz Ruszkowski5Miroslaw Gilski6Mariusz Jaskolski7Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandDepartment of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandThe ReAV enzyme from <i>Rhizobium etli</i>, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism.https://www.mdpi.com/2218-273X/15/7/944hydrolaseamidohydrolaseL-asparaginaseleukemiametalloproteinsite-directed mutagenesis |
| spellingShingle | Kinga Pokrywka Marta Grzechowiak Joanna Sliwiak Paulina Worsztynowicz Joanna I. Loch Milosz Ruszkowski Miroslaw Gilski Mariusz Jaskolski Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV Biomolecules hydrolase amidohydrolase L-asparaginase leukemia metalloprotein site-directed mutagenesis |
| title | Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV |
| title_full | Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV |
| title_fullStr | Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV |
| title_full_unstemmed | Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV |
| title_short | Probing the Active Site of Class 3 L-Asparaginase by Mutagenesis: Mutations of the Ser-Lys Tandems of ReAV |
| title_sort | probing the active site of class 3 l asparaginase by mutagenesis mutations of the ser lys tandems of reav |
| topic | hydrolase amidohydrolase L-asparaginase leukemia metalloprotein site-directed mutagenesis |
| url | https://www.mdpi.com/2218-273X/15/7/944 |
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