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Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli
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| Zeitschriftentitel: | Proceedings of the National Academy of Sciences |
|---|---|
| Personen und Körperschaften: | |
| In: | Proceedings of the National Academy of Sciences, 94, 1997, 12, S. 6553-6558 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Proceedings of the National Academy of Sciences
|
| Schlagwörter: |
| author_facet |
Lange, Theo Lange, Theo |
|---|---|
| author |
Lange, Theo |
| spellingShingle |
Lange, Theo Proceedings of the National Academy of Sciences Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli Multidisciplinary |
| author_sort |
lange, theo |
| spelling |
Lange, Theo 0027-8424 1091-6490 Proceedings of the National Academy of Sciences Multidisciplinary http://dx.doi.org/10.1073/pnas.94.12.6553 <jats:p> Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant <jats:italic>Escherichia coli</jats:italic> . <jats:italic>In vitro</jats:italic> translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOS <jats:italic>Elox</jats:italic> . An aliquot of the amplified library was converted to plasmids <jats:italic>in vivo</jats:italic> and used for transformation of <jats:italic>E. coli</jats:italic> BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated <jats:italic>M</jats:italic> <jats:sub>r</jats:sub> of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA <jats:sub>12</jats:sub> -aldehyde to GA <jats:sub>12</jats:sub> and GA <jats:sub>14</jats:sub> -aldehyde to GA <jats:sub>14</jats:sub> . Evidence was obtained for further metabolism of GA <jats:sub>12</jats:sub> by the 7-oxidase to four products, two of which are monohydroxylated GA <jats:sub>12</jats:sub> . The ORF of the 20-oxidase is—apart from seven changes, resulting in four amino acid substitutions—identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties. </jats:p> Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in <i>Escherichia coli</i> Proceedings of the National Academy of Sciences |
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Proceedings of the National Academy of Sciences, 1997 |
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Proceedings of the National Academy of Sciences, 1997 |
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1997 |
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Proceedings of the National Academy of Sciences |
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Proceedings of the National Academy of Sciences |
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49 |
| title |
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_unstemmed |
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_full |
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_fullStr |
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_full_unstemmed |
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_short |
Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_sort |
cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in
<i>escherichia coli</i> |
| topic |
Multidisciplinary |
| url |
http://dx.doi.org/10.1073/pnas.94.12.6553 |
| publishDate |
1997 |
| physical |
6553-6558 |
| description |
<jats:p>
Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant
<jats:italic>Escherichia coli</jats:italic>
.
<jats:italic>In vitro</jats:italic>
translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOS
<jats:italic>Elox</jats:italic>
. An aliquot of the amplified library was converted to plasmids
<jats:italic>in vivo</jats:italic>
and used for transformation of
<jats:italic>E. coli</jats:italic>
BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated
<jats:italic>M</jats:italic>
<jats:sub>r</jats:sub>
of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA
<jats:sub>12</jats:sub>
-aldehyde to GA
<jats:sub>12</jats:sub>
and GA
<jats:sub>14</jats:sub>
-aldehyde to GA
<jats:sub>14</jats:sub>
. Evidence was obtained for further metabolism of GA
<jats:sub>12</jats:sub>
by the 7-oxidase to four products, two of which are monohydroxylated GA
<jats:sub>12</jats:sub>
. The ORF of the 20-oxidase is—apart from seven changes, resulting in four amino acid substitutions—identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties.
</jats:p> |
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| container_title | Proceedings of the National Academy of Sciences |
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| description | <jats:p> Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant <jats:italic>Escherichia coli</jats:italic> . <jats:italic>In vitro</jats:italic> translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOS <jats:italic>Elox</jats:italic> . An aliquot of the amplified library was converted to plasmids <jats:italic>in vivo</jats:italic> and used for transformation of <jats:italic>E. coli</jats:italic> BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated <jats:italic>M</jats:italic> <jats:sub>r</jats:sub> of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA <jats:sub>12</jats:sub> -aldehyde to GA <jats:sub>12</jats:sub> and GA <jats:sub>14</jats:sub> -aldehyde to GA <jats:sub>14</jats:sub> . Evidence was obtained for further metabolism of GA <jats:sub>12</jats:sub> by the 7-oxidase to four products, two of which are monohydroxylated GA <jats:sub>12</jats:sub> . The ORF of the 20-oxidase is—apart from seven changes, resulting in four amino acid substitutions—identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties. </jats:p> |
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| imprint | Proceedings of the National Academy of Sciences, 1997 |
| imprint_str_mv | Proceedings of the National Academy of Sciences, 1997 |
| institution | DE-Brt1, DE-D161, DE-Zwi2, DE-Gla1, DE-Zi4, DE-15, DE-Pl11, DE-Rs1, DE-105, DE-14, DE-Ch1, DE-L229, DE-D275, DE-Bn3 |
| issn | 1091-6490, 0027-8424 |
| issn_str_mv | 1091-6490, 0027-8424 |
| language | English |
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| mega_collection | Proceedings of the National Academy of Sciences (CrossRef) |
| physical | 6553-6558 |
| publishDate | 1997 |
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| publisher | Proceedings of the National Academy of Sciences |
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| recordtype | ai |
| series | Proceedings of the National Academy of Sciences |
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| spelling | Lange, Theo 0027-8424 1091-6490 Proceedings of the National Academy of Sciences Multidisciplinary http://dx.doi.org/10.1073/pnas.94.12.6553 <jats:p> Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant <jats:italic>Escherichia coli</jats:italic> . <jats:italic>In vitro</jats:italic> translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOS <jats:italic>Elox</jats:italic> . An aliquot of the amplified library was converted to plasmids <jats:italic>in vivo</jats:italic> and used for transformation of <jats:italic>E. coli</jats:italic> BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated <jats:italic>M</jats:italic> <jats:sub>r</jats:sub> of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA <jats:sub>12</jats:sub> -aldehyde to GA <jats:sub>12</jats:sub> and GA <jats:sub>14</jats:sub> -aldehyde to GA <jats:sub>14</jats:sub> . Evidence was obtained for further metabolism of GA <jats:sub>12</jats:sub> by the 7-oxidase to four products, two of which are monohydroxylated GA <jats:sub>12</jats:sub> . The ORF of the 20-oxidase is—apart from seven changes, resulting in four amino acid substitutions—identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties. </jats:p> Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in <i>Escherichia coli</i> Proceedings of the National Academy of Sciences |
| spellingShingle | Lange, Theo, Proceedings of the National Academy of Sciences, Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli, Multidisciplinary |
| title | Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_full | Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_fullStr | Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_full_unstemmed | Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_short | Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| title_sort | cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in <i>escherichia coli</i> |
| title_unstemmed | Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli |
| topic | Multidisciplinary |
| url | http://dx.doi.org/10.1073/pnas.94.12.6553 |