In vitro reconstituted biotransformation of 4-fluorothreonine from fluoride ion: application of the fluorinase.
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 flA and flB genes of the fluorometabolite gene cluster in S. cattleya, were overexpressed. Also, an isomerase gene product that can convert 5-FDRP 6 to 5-FDRibulP 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.
Deng
Hai
H
School of Chemistry and Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, UK.
Cross
Stuart M
SM
McGlinchey
Ryan P
RP
Hamilton
John T G
JT
O'Hagan
David
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D005459:Fluorides / Q000737:chemistry* / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013302:Streptomyces / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013912:Threonine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014153:Transaldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D001426:Bacterial Proteins; C048271:4-fluorothreonine; D013912:Threonine; D010088:Oxidoreductases; C495766:fluorinase; D014153:Transaldolase; D005634:Fructose-Bisphosphate Aldolase; D005459:Fluorides