Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i>
The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step “hydantoinase process” based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from <i>Pseudomonas</...
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2024-10-01
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| author | Marina Paronyan Haykanush Koloyan Hovsep Aganyants Artur Hambardzumyan Tigran Soghomonyan Sona Avetisyan Sergey Kocharov Henry Panosyan Vehary Sakanyan Anichka Hovsepyan |
| author_facet | Marina Paronyan Haykanush Koloyan Hovsep Aganyants Artur Hambardzumyan Tigran Soghomonyan Sona Avetisyan Sergey Kocharov Henry Panosyan Vehary Sakanyan Anichka Hovsepyan |
| author_sort | Marina Paronyan |
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| description | The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step “hydantoinase process” based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from <i>Pseudomonas</i>, the encoded gene of which was chemically synthesized and cloned into <i>Escherichia coli</i>. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones. |
| format | Article |
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| institution | OA Journals |
| issn | 2673-6284 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | MDPI AG |
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| spelling | doaj-art-e7d464198c0e425da3007555279e3d3c2025-08-20T02:01:06ZengMDPI AGBioTech2673-62842024-10-011344010.3390/biotech13040040Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i>Marina Paronyan0Haykanush Koloyan1Hovsep Aganyants2Artur Hambardzumyan3Tigran Soghomonyan4Sona Avetisyan5Sergey Kocharov6Henry Panosyan7Vehary Sakanyan8Anichka Hovsepyan9Scientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaScientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaScientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaScientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaScientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaScientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaScientific Technological Centre of Organic and Pharmaceutical Chemistry SNPO, National Academy of Sciences of Armenia, Yerevan 0014, ArmeniaScientific Technological Centre of Organic and Pharmaceutical Chemistry SNPO, National Academy of Sciences of Armenia, Yerevan 0014, ArmeniaFaculty of Science and Technique, Nantes University, 44035 Nantes, FranceScientific and Production Center ”Armbiotechnology”, National Academy of Sciences of Armenia, Yerevan 0056, ArmeniaThe synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step “hydantoinase process” based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from <i>Pseudomonas</i>, the encoded gene of which was chemically synthesized and cloned into <i>Escherichia coli</i>. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones.https://www.mdpi.com/2673-6284/13/4/40D-carbamoylasehomology modelingmolecular dockingmolecular dynamicsN-carbamoyl-D-alanineN-carbamoyl-D-tryptophan |
| spellingShingle | Marina Paronyan Haykanush Koloyan Hovsep Aganyants Artur Hambardzumyan Tigran Soghomonyan Sona Avetisyan Sergey Kocharov Henry Panosyan Vehary Sakanyan Anichka Hovsepyan Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i> BioTech D-carbamoylase homology modeling molecular docking molecular dynamics N-carbamoyl-D-alanine N-carbamoyl-D-tryptophan |
| title | Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i> |
| title_full | Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i> |
| title_fullStr | Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i> |
| title_full_unstemmed | Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i> |
| title_short | Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i> |
| title_sort | structural analysis and substrate specificity of d carbamoylase from i pseudomonas i |
| topic | D-carbamoylase homology modeling molecular docking molecular dynamics N-carbamoyl-D-alanine N-carbamoyl-D-tryptophan |
| url | https://www.mdpi.com/2673-6284/13/4/40 |
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