Epoxide formation on the aromatic B ring of flavanone by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes KF707

Abstract

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2' and C3' on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2',3'-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.

FIG. 1.

Pathways for the metabolic initiation of arene ring structures by prokaryotic and eukaryotic oxygenases. (Adapted from reference 44 with the permission of the author and the publisher.)

FIG. 2.

Organization of biphenyl dioxygenase of P. pseudoalcaligenes KF707.

FIG. 3.

Structures and nomenclatures of flavone and flavanone.

FIG. 4.

HPLC chromatograms of biotransformation products produced from 2(R)- or 2(S)-flavanone by whole cells of E. coli expressing biphenyl dioxygenase of P. pseudoalcaligenes KF707. Right inset of each chromatogram are the UV spectra for oxidation intermediates I and II.

FIG. 5.

CD spectra of 2(R)- (cut line) and 2(S)- (solid line) flavanone.

FIG. 6.

Proposed reaction mechanism of flavone (A) and flavanone (B) oxidation by biphenyl dioxygenase. The structures were produced from Molecular Mechanics Calculation using HyperChem, and the X-ray crystallographic structures of flavone and flavanones (CCDC) were approximately the same as the calculated ones.

FIG. 7.

Biotransformation kinetics of 2(R)-flavanone (A) and 2(S)-flavanone (B) to the corresponding flavanone epoxides by whole cells of E. coli expressing biphenyl dioxygenase of P. pseudoalcaligenes KF707. Symbols: ○, flavanone; •, flavanone epoxide.