In vitro reconstituted biotransformation of 4-fluorothreonine from fluoride ion: Application of the fluorinase

Hai Deng, Stuart M. Cross, Ryan P. McGlinchey, John T.G. Hamilton, David O'Hagan

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

In this paper, we report that fluoride ion is converted to the amino acid/antibiotic 4-fluorothreonine 2 in a biotransformation involving five (steps a-e) overexpressed enzymes. The biotransformation validates the biosynthetic pathway to 4-fluorothreonine in the bacterium Streptomyces cattleya (Schaffrath et al., 2002). To achieve an in vitro biotransformation, the fluorinase and the purine nucleoside phosphorylase (PNP) enzymes (steps a and b), which are coded for by the fIA and fIB genes of the fluorometabolite gene cluster in S. cattleya, were overexpressed. Also, an isomerase gene product that can convert 5-FDRP 6 to 5-FDRibuIP 7 (step c) was identified in S. cattleya, and the enzyme was overexpressed for the biotransformation. A fuculose aldolase gene from S. coelicolor was overexpressed in E, coli and was used as a surrogate aldolase (step d) in these experiments. To complete the complement of enzymes, an ORF coding the PLP-dependent transaldolase, the final enzyme of the fluorometabolite pathway, was identified in genomic DNA by a reverse genetics approach, and the S. cattleya gene/enzyme was then overexpressed in S. lividans. This latter enzyme is an unusual PLP-dependent catalyst with some homology to both bacterial serine hydroxymethyl transferases (SHMT) and C5 sugar isomerases/epimerases. The biotransformation demonstrates the power of the fluorinase to initiate C-F bond formation for organo-fluorine synthesis.
Original languageEnglish
Pages (from-to)1268-1276
Number of pages9
JournalChemistry & Biology
Volume15
Issue number12
Early online date10 Nov 2008
DOIs
Publication statusPublished - 22 Dec 2008

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Biotransformation
Fluorides
Ions
Genes
Enzymes
Isomerases
Fructose-Bisphosphate Aldolase
Transaldolase
Hydroxymethyl and Formyl Transferases
Purine-Nucleoside Phosphorylase
Racemases and Epimerases
Reverse Genetics
Fluorine
Biosynthetic Pathways
Streptomyces
Multigene Family
fluorinase
In Vitro Techniques
4-fluorothreonine
Sugars

Keywords

  • 5-methylthioribose 1-phosphate isomerase
  • streptomyces-cattleya
  • fluorometabolite biosynthesis
  • crystal-structure
  • L-ribulose-5-phosphate 4-epimerase
  • bacillus-subtilis
  • bond formation
  • gene-cluster
  • amino-acids
  • identification

Cite this

In vitro reconstituted biotransformation of 4-fluorothreonine from fluoride ion : Application of the fluorinase. / Deng, Hai; Cross, Stuart M.; McGlinchey, Ryan P.; Hamilton, John T.G.; O'Hagan, David.

In: Chemistry & Biology, Vol. 15, No. 12, 22.12.2008, p. 1268-1276.

Research output: Contribution to journalArticle

Deng, Hai ; Cross, Stuart M. ; McGlinchey, Ryan P. ; Hamilton, John T.G. ; O'Hagan, David. / In vitro reconstituted biotransformation of 4-fluorothreonine from fluoride ion : Application of the fluorinase. In: Chemistry & Biology. 2008 ; Vol. 15, No. 12. pp. 1268-1276.
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AB - In this paper, we report that fluoride ion is converted to the amino acid/antibiotic 4-fluorothreonine 2 in a biotransformation involving five (steps a-e) overexpressed enzymes. The biotransformation validates the biosynthetic pathway to 4-fluorothreonine in the bacterium Streptomyces cattleya (Schaffrath et al., 2002). To achieve an in vitro biotransformation, the fluorinase and the purine nucleoside phosphorylase (PNP) enzymes (steps a and b), which are coded for by the fIA and fIB genes of the fluorometabolite gene cluster in S. cattleya, were overexpressed. Also, an isomerase gene product that can convert 5-FDRP 6 to 5-FDRibuIP 7 (step c) was identified in S. cattleya, and the enzyme was overexpressed for the biotransformation. A fuculose aldolase gene from S. coelicolor was overexpressed in E, coli and was used as a surrogate aldolase (step d) in these experiments. To complete the complement of enzymes, an ORF coding the PLP-dependent transaldolase, the final enzyme of the fluorometabolite pathway, was identified in genomic DNA by a reverse genetics approach, and the S. cattleya gene/enzyme was then overexpressed in S. lividans. This latter enzyme is an unusual PLP-dependent catalyst with some homology to both bacterial serine hydroxymethyl transferases (SHMT) and C5 sugar isomerases/epimerases. The biotransformation demonstrates the power of the fluorinase to initiate C-F bond formation for organo-fluorine synthesis.

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KW - amino-acids

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JO - Chemistry & Biology

JF - Chemistry & Biology

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