Marina Paronyan, Haykanush Koloyan, Hovsep Aganyants, Artur Hambardzumyan, Tigran Soghomonyan, Sona Avetisyan, Sergey Kocharov, Henry Panosyan, Vehary Sakanyan, Anichka Hovsepyan
{"title":"假单胞菌 D-氨基甲酰酶的结构分析和底物特异性","authors":"Marina Paronyan, Haykanush Koloyan, Hovsep Aganyants, Artur Hambardzumyan, Tigran Soghomonyan, Sona Avetisyan, Sergey Kocharov, Henry Panosyan, Vehary Sakanyan, Anichka Hovsepyan","doi":"10.3390/biotech13040040","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":"13 4","pages":""},"PeriodicalIF":2.7000,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11503299/pdf/","citationCount":"0","resultStr":"{\"title\":\"Structural Analysis and Substrate Specificity of D-Carbamoylase from <i>Pseudomonas</i>.\",\"authors\":\"Marina Paronyan, Haykanush Koloyan, Hovsep Aganyants, Artur Hambardzumyan, Tigran Soghomonyan, Sona Avetisyan, Sergey Kocharov, Henry Panosyan, Vehary Sakanyan, Anichka Hovsepyan\",\"doi\":\"10.3390/biotech13040040\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":34490,\"journal\":{\"name\":\"BioTech\",\"volume\":\"13 4\",\"pages\":\"\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2024-10-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11503299/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"BioTech\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3390/biotech13040040\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioTech","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/biotech13040040","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Structural Analysis and Substrate Specificity of D-Carbamoylase from Pseudomonas.
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 Pseudomonas, the encoded gene of which was chemically synthesized and cloned into Escherichia coli. 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.