Cis-dihydrodiol production on flavone B-ring by biphenyl dioxygenase from pseudomonas pseudoalcaligenes KF707 expressed in escherichia coli

Abstract

Escherichia coli JM109 expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Metabolites from flavone and 5,7-dihydroxyflavone were shown on the HPLC chromatogram, which were not found in the control experiments. However, 6,7-, 7,8-, 2¡¯,3¡¯-, 3¡¯,4¡¯-, 5,4¡¯- dihydroxyflavone, 5,6,7-trihydroxyflavone, 5-hydroxy -7-methoxy flavone, and 8- hydroxy-7-methoxyflavone were not catalyzed by both enzymes. The maximum UVvisible absorbance for the metabolites from flavone and 5,7-dihydroxyflavone was found at 337 and 348 nm respectively by the photodiode array detector equipped in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed 257 and 289 m/z [M + H]+ for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis of the 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2¡¯, and C3¡¯ on the B-ring of flavone, resulting in the formation of flavone cisdihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since the product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase able to synthesize noble compounds from plant secondary metabolites.