Doris del Carmen Fuentes , Lucía Soto-Urzua , Lino Javier Martínez-Soto , Luis Javier Martínez-Morales
{"title":"来自 Azospirillum baldaniorum 的 PHB 聚合酶(PhbC)的生物信息学和功能分析","authors":"Doris del Carmen Fuentes , Lucía Soto-Urzua , Lino Javier Martínez-Soto , Luis Javier Martínez-Morales","doi":"10.1016/j.jgeb.2024.100403","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><p><em>Azospirillum baldaniorum</em> Sp245 produces poly-β-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-β-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release.</p></div><div><h3>Methods</h3><p>A study was conducted using <em>in silico</em> analysis of the two <em>phb</em>C genes of <em>A. baldaniorum</em> Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO® vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from <em>Azospirillum baldaniorum</em> (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV–Vis assay was performed. To confirm the presence of the poly-β-hydroxybutyrate product, an NMR assay was performed.</p></div><div><h3>Results</h3><p><em>In silico</em> analyses, it is revealed that AbPhbC1 and the PhbC2 protein from <em>Azospirillum baldaniorum</em> (AbPhbC2) retain the poly-β-hydroxybutyrate polymerase and α/β hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four polyβ-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different; in AbPhbC1, it is in the closed form; in AbPhbC2, it is in the open form; and in AbPhbC2, it lacks the EC region as class III and IV poly-β-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV–Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and commercial poly-β-hydroxybutyrate samples suggested their presence.</p></div><div><h3>Conclusion</h3><p><em>In silico</em> analyses suggested that AbPhbC1 and AbPhbC2 are structurally functional, except that AbPhbC2 might require the PhaR subunit for its activity; this strongly suggests that it could be a class IV poly-β-hydroxyalcanoate synthase. UV–Vis scanning and NMR spectroscopy revealed the synthesis of poly-β-hydroxybutyrate by the <em>A. baldaniorum</em> enzyme AbPhbC1, indicating that the enzyme is functional.</p></div>","PeriodicalId":53463,"journal":{"name":"Journal of Genetic Engineering and Biotechnology","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1687157X24001069/pdfft?md5=afe7ad1f4b87cee05942aeb268459c3e&pid=1-s2.0-S1687157X24001069-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Bioinformatic and functional analysis of a PHB polymerase (PhbC) from Azospirillum baldaniorum\",\"authors\":\"Doris del Carmen Fuentes , Lucía Soto-Urzua , Lino Javier Martínez-Soto , Luis Javier Martínez-Morales\",\"doi\":\"10.1016/j.jgeb.2024.100403\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><p><em>Azospirillum baldaniorum</em> Sp245 produces poly-β-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-β-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release.</p></div><div><h3>Methods</h3><p>A study was conducted using <em>in silico</em> analysis of the two <em>phb</em>C genes of <em>A. baldaniorum</em> Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO® vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from <em>Azospirillum baldaniorum</em> (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV–Vis assay was performed. To confirm the presence of the poly-β-hydroxybutyrate product, an NMR assay was performed.</p></div><div><h3>Results</h3><p><em>In silico</em> analyses, it is revealed that AbPhbC1 and the PhbC2 protein from <em>Azospirillum baldaniorum</em> (AbPhbC2) retain the poly-β-hydroxybutyrate polymerase and α/β hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four polyβ-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different; in AbPhbC1, it is in the closed form; in AbPhbC2, it is in the open form; and in AbPhbC2, it lacks the EC region as class III and IV poly-β-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV–Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and commercial poly-β-hydroxybutyrate samples suggested their presence.</p></div><div><h3>Conclusion</h3><p><em>In silico</em> analyses suggested that AbPhbC1 and AbPhbC2 are structurally functional, except that AbPhbC2 might require the PhaR subunit for its activity; this strongly suggests that it could be a class IV poly-β-hydroxyalcanoate synthase. UV–Vis scanning and NMR spectroscopy revealed the synthesis of poly-β-hydroxybutyrate by the <em>A. baldaniorum</em> enzyme AbPhbC1, indicating that the enzyme is functional.</p></div>\",\"PeriodicalId\":53463,\"journal\":{\"name\":\"Journal of Genetic Engineering and Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-07-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S1687157X24001069/pdfft?md5=afe7ad1f4b87cee05942aeb268459c3e&pid=1-s2.0-S1687157X24001069-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Genetic Engineering and Biotechnology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1687157X24001069\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Genetic Engineering and Biotechnology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1687157X24001069","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
Bioinformatic and functional analysis of a PHB polymerase (PhbC) from Azospirillum baldaniorum
Background
Azospirillum baldaniorum Sp245 produces poly-β-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-β-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release.
Methods
A study was conducted using in silico analysis of the two phbC genes of A. baldaniorum Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO® vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from Azospirillum baldaniorum (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV–Vis assay was performed. To confirm the presence of the poly-β-hydroxybutyrate product, an NMR assay was performed.
Results
In silico analyses, it is revealed that AbPhbC1 and the PhbC2 protein from Azospirillum baldaniorum (AbPhbC2) retain the poly-β-hydroxybutyrate polymerase and α/β hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four polyβ-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different; in AbPhbC1, it is in the closed form; in AbPhbC2, it is in the open form; and in AbPhbC2, it lacks the EC region as class III and IV poly-β-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV–Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and commercial poly-β-hydroxybutyrate samples suggested their presence.
Conclusion
In silico analyses suggested that AbPhbC1 and AbPhbC2 are structurally functional, except that AbPhbC2 might require the PhaR subunit for its activity; this strongly suggests that it could be a class IV poly-β-hydroxyalcanoate synthase. UV–Vis scanning and NMR spectroscopy revealed the synthesis of poly-β-hydroxybutyrate by the A. baldaniorum enzyme AbPhbC1, indicating that the enzyme is functional.
期刊介绍:
Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts