Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc
L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2024-02-01
|
| Series: | Frontiers in Chemistry |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fchem.2024.1373312/full |
| _version_ | 1797300417529380864 |
|---|---|
| author | Joanna Sliwiak Paulina Worsztynowicz Kinga Pokrywka Joanna I. Loch Marta Grzechowiak Mariusz Jaskolski Mariusz Jaskolski |
| author_facet | Joanna Sliwiak Paulina Worsztynowicz Kinga Pokrywka Joanna I. Loch Marta Grzechowiak Mariusz Jaskolski Mariusz Jaskolski |
| author_sort | Joanna Sliwiak |
| collection | DOAJ |
| description | L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme. |
| first_indexed | 2024-03-07T23:06:52Z |
| format | Article |
| id | doaj.art-3a0d808633184a3cb22ff775949393da |
| institution | Directory Open Access Journal |
| issn | 2296-2646 |
| language | English |
| last_indexed | 2024-03-07T23:06:52Z |
| publishDate | 2024-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Chemistry |
| spelling | doaj.art-3a0d808633184a3cb22ff775949393da2024-02-22T04:34:18ZengFrontiers Media S.A.Frontiers in Chemistry2296-26462024-02-011210.3389/fchem.2024.13733121373312Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zincJoanna Sliwiak0Paulina Worsztynowicz1Kinga Pokrywka2Joanna I. Loch3Marta Grzechowiak4Mariusz Jaskolski5Mariusz Jaskolski6Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, PolandDepartment of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Krakow, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, PolandDepartment of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, Poznan, PolandL-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.https://www.frontiersin.org/articles/10.3389/fchem.2024.1373312/fullClass 3 L-asparaginaseReAVenzymatic mechanismamidohydrolaseenzyme kineticsRhizobium etli |
| spellingShingle | Joanna Sliwiak Paulina Worsztynowicz Kinga Pokrywka Joanna I. Loch Marta Grzechowiak Mariusz Jaskolski Mariusz Jaskolski Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc Frontiers in Chemistry Class 3 L-asparaginase ReAV enzymatic mechanism amidohydrolase enzyme kinetics Rhizobium etli |
| title | Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc |
| title_full | Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc |
| title_fullStr | Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc |
| title_full_unstemmed | Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc |
| title_short | Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc |
| title_sort | biochemical characterization of l asparaginase isoforms from rhizobium etli the boosting effect of zinc |
| topic | Class 3 L-asparaginase ReAV enzymatic mechanism amidohydrolase enzyme kinetics Rhizobium etli |
| url | https://www.frontiersin.org/articles/10.3389/fchem.2024.1373312/full |
| work_keys_str_mv | AT joannasliwiak biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc AT paulinaworsztynowicz biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc AT kingapokrywka biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc AT joannailoch biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc AT martagrzechowiak biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc AT mariuszjaskolski biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc AT mariuszjaskolski biochemicalcharacterizationoflasparaginaseisoformsfromrhizobiumetlitheboostingeffectofzinc |