Synthesis of [(14)C]pyrroloquinoline quinone (PQQ) in E. coli using genes for PQQ synthesis from K. pneumoniae.
Radiochemical forms of pyrroloquinoline quinone (PQQ) are of utility in studies to determine the metabolic role and fate of PQQ in biological systems. Accordingly, we have synthesized [(14)C]PQQ using a tyrosine auxotrophic strain of Escherichia coli (AT2471). A construct containing the six genes required for PQQ synthesis from Klebsiella pneumoniae was used to transform the auxotrophic strain of E. coli. E. coli were then grown in minimal M9 medium containing 3.7x10(9) Bq/mmol [(14)C]tyrosine. At confluence, the medium was collected and applied to a DEAE A-25 anionic exchange column; [(14)C]PQQ was eluted using a KCl gradient (0-2 M in 0.1 M potassium phosphate buffer, pH 7.0). Radioactivity co-eluting as PQQ was next pooled, acidified and passed through a C-18 column; [(14)C]PQQ was eluted with a phosphate buffer (0.1 M, pH 7.0). Reverse phase HPLC (C-18) using either the ion-pairing agent trifluoroacetic acid (0. 1%) and an acetonitrile gradient or phosphoric acid and a methanol gradient were used to isolate [(14)C]PQQ. Fractions were collected and analyzed by liquid scintillation counting. (14)C-labelled compounds isolated from the medium eluted corresponding to the elution of various tyrosine-derived products or PQQ. The radioactive compound corresponding to PQQ was also reacted with acetone to form 5-acetonyl-PQQ, which co-eluted with a 5-acetonyl-PQQ standard, as a validation of [(14)C]PQQ synthesis. The specific activity of synthesized [(14)C]PQQ was 3.7x10(9) Bq/mmol [(14)C]PQQ, equal to that of [U-(14)C]tyrosine initially added to the medium.
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Department of Nutrition, One Shields Avenue, University of California, Davis, Davis, CA 95616, USA.
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D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000378:metabolism; D045542:PQQ Cofactor; D010957:Plasmids; D015363:Quinolones / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism*
D002250:Carbon Radioisotopes; D003067:Coenzymes; D015363:Quinolones; D011809:Quinones; D045542:PQQ Cofactor