Articles
PURIFICATION AND CHARACTERIZATION OF NOVEL ENZYMES IN TERPENOID BIOSYNTHESIS
Article number
677_15
Pages
115 – 122
Language
English
Abstract
In this study, enzymes catalyzing hitherto steps of the alternative terpenoid biosynthesis were isolated and characterized.
The genes specifying these enzymes were found by a bioinformatic approach using complete sequenced genomes of Escherichia coli. By genomic analysis, the putative ygbP, ychB and ygbB orthologs distribute in line with orthologs of dxs and dxr genes, which were already shown to be involved in the non-mevalonate pathway.
Therefore, they were cloned and overexpressed.
The enzymes were purified to homogeneity from recombinant Escherichia coli strains by column chromatography.
Convenient assay methods for the measurement of enzyme activities were established.
The enzymes were characterized in detail.
The recombinant YgbP enzyme was shown to catalyze the conversion of 2C-methyl-D-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-D-erythritol by reaction with CTP. Then, the recombinant YchB enzyme catalyzes the phosphorylation at the position 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP dependent reaction, formed 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate.
Finally, the recombinant YgbB enzyme converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate under release of CMP. Recently, the ygbP, ychB and ygbB genes were renamed properly to ispD, ispE and IspF respectively.
The purified recombinant enzymes were used as tools for the preparation of large amount of intermediates in order to elucidate the later steps leading to isopentenyl diphosphate.
Moreover, understanding of those enzyme properties can be applied to improve biosynthesis of pharmacologically terpenoid compounds in microorganisms and plants, which is the perspective approach in natural product chemistry.
The genes specifying these enzymes were found by a bioinformatic approach using complete sequenced genomes of Escherichia coli. By genomic analysis, the putative ygbP, ychB and ygbB orthologs distribute in line with orthologs of dxs and dxr genes, which were already shown to be involved in the non-mevalonate pathway.
Therefore, they were cloned and overexpressed.
The enzymes were purified to homogeneity from recombinant Escherichia coli strains by column chromatography.
Convenient assay methods for the measurement of enzyme activities were established.
The enzymes were characterized in detail.
The recombinant YgbP enzyme was shown to catalyze the conversion of 2C-methyl-D-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-D-erythritol by reaction with CTP. Then, the recombinant YchB enzyme catalyzes the phosphorylation at the position 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol in an ATP dependent reaction, formed 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate.
Finally, the recombinant YgbB enzyme converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate under release of CMP. Recently, the ygbP, ychB and ygbB genes were renamed properly to ispD, ispE and IspF respectively.
The purified recombinant enzymes were used as tools for the preparation of large amount of intermediates in order to elucidate the later steps leading to isopentenyl diphosphate.
Moreover, understanding of those enzyme properties can be applied to improve biosynthesis of pharmacologically terpenoid compounds in microorganisms and plants, which is the perspective approach in natural product chemistry.
Publication
Authors
J. Wungsintaweekul, S. Sagner, M. Fellermeier, M.H. Zenk, F. Rohdich, K. Kis, C.A. Schuhr, S. Hecht, A. Bacher
Keywords
non-mevalonate, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, Escherichia coli
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