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Recombinant AroL-Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3-Phosphate
- Source :
- Biotechnology journal. 13(8)
- Publication Year :
- 2017
-
Abstract
- Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate is only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli. The time course of the shikimate kinase-catalyzed phosphorylation is investigated by 1 H- and 31 P-NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3-phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3-phosphate, with high HPLC- and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3-phosphate is superior to any other method and has been successfully scaled up to multi-gram scale.
- Subjects :
- 0301 basic medicine
Shikimic Acid
010402 general chemistry
01 natural sciences
Applied Microbiology and Biotechnology
Shikimate kinase
Cofactor
03 medical and health sciences
chemistry.chemical_compound
Enzyme Stability
Escherichia coli
Shikimate pathway
Phosphorylation
chemistry.chemical_classification
ATP synthase
biology
Escherichia coli Proteins
General Medicine
Shikimic acid
Recombinant Proteins
0104 chemical sciences
Phosphotransferases (Alcohol Group Acceptor)
030104 developmental biology
Enzyme
chemistry
Biochemistry
biology.protein
Molecular Medicine
Phosphoenolpyruvate carboxykinase
Pyruvate kinase
Subjects
Details
- ISSN :
- 18607314
- Volume :
- 13
- Issue :
- 8
- Database :
- OpenAIRE
- Journal :
- Biotechnology journal
- Accession number :
- edsair.doi.dedup.....1f4b0ea5fa224e05b789efac13696793