Ute Neudert , Isabel M Martı́nez-Férez , Paul D Fraser , Gerhard Sandmann
{"title":"一种活性植物烯合成酶的表达及酶的生化特性","authors":"Ute Neudert , Isabel M Martı́nez-Férez , Paul D Fraser , Gerhard Sandmann","doi":"10.1016/S0005-2760(98)00017-4","DOIUrl":null,"url":null,"abstract":"<div><p>The <em>crtB</em> gene encoding phytoene synthase from the carotenogenic enterobacterium <em>Erwinia uredovora</em> was overexpressed to about 20% of the total cellular protein in <em>Escherichia coli</em>. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in <em>E. coli</em>, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes <em>crtE</em> and <em>idi</em>, encoding the <em>Erwinia</em> GGPP synthase and <em>Rhodobacter</em> isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15-<em>cis</em> and all-<em>trans</em> isomeric configuration. The essential cofactors required were ATP in combinations with either Mn<sup>2+</sup> or Mg<sup>2+</sup>. The <em>K</em><sub>m</sub> value for GGPP was determined as 41 <em>μ</em>M. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The <em>I</em><sub>50</sub> value for the latter inhibitor was 15 <em>μ</em>M. Lineweaver–Burk plots showed constant <em>K</em><sub>m</sub> values in the presence or absence of squalestatin.</p></div>","PeriodicalId":100162,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1998-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0005-2760(98)00017-4","citationCount":"34","resultStr":"{\"title\":\"Expression of an active phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme\",\"authors\":\"Ute Neudert , Isabel M Martı́nez-Férez , Paul D Fraser , Gerhard Sandmann\",\"doi\":\"10.1016/S0005-2760(98)00017-4\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The <em>crtB</em> gene encoding phytoene synthase from the carotenogenic enterobacterium <em>Erwinia uredovora</em> was overexpressed to about 20% of the total cellular protein in <em>Escherichia coli</em>. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in <em>E. coli</em>, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes <em>crtE</em> and <em>idi</em>, encoding the <em>Erwinia</em> GGPP synthase and <em>Rhodobacter</em> isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15-<em>cis</em> and all-<em>trans</em> isomeric configuration. The essential cofactors required were ATP in combinations with either Mn<sup>2+</sup> or Mg<sup>2+</sup>. The <em>K</em><sub>m</sub> value for GGPP was determined as 41 <em>μ</em>M. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The <em>I</em><sub>50</sub> value for the latter inhibitor was 15 <em>μ</em>M. Lineweaver–Burk plots showed constant <em>K</em><sub>m</sub> values in the presence or absence of squalestatin.</p></div>\",\"PeriodicalId\":100162,\"journal\":{\"name\":\"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1998-05-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/S0005-2760(98)00017-4\",\"citationCount\":\"34\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0005276098000174\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0005276098000174","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Expression of an active phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme
The crtB gene encoding phytoene synthase from the carotenogenic enterobacterium Erwinia uredovora was overexpressed to about 20% of the total cellular protein in Escherichia coli. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in E. coli, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes crtE and idi, encoding the Erwinia GGPP synthase and Rhodobacter isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15-cis and all-trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn2+ or Mg2+. The Km value for GGPP was determined as 41 μM. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The I50 value for the latter inhibitor was 15 μM. Lineweaver–Burk plots showed constant Km values in the presence or absence of squalestatin.