Protein engineering on biphenyl dioxygenase for conferring activity to convert 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin).
A central part (amino-acid position 268-397 of 458 amino-acid residues) of the biphenyl dioxygenase large (alpha) subunit, BphA1, from Pseudomonas pseudoalcaligenes strain KF707 was exchanged with the corresponding part of BphA1 from another biphenyl-degrading bacterium, Pseudomonas putida strain KF715, to construct hybrid BphA1, BphA1 (715-707). When expressed in Escherichia coli together with the bphA2A3A4BC genes from strain KF707, this enzyme was shown to possess activity for degrading both 1-phenylnaphthalene and 2-phenylnaphthalene. Between central parts of BphA1 from strains KF707 and KF715, the difference of amino-acid residues resided only in position 324-325. An attempt was made to improve the substrate preference of BphA1 by applying random amino-acid substitutions at these positions to BphA1 (715-707). After screening the mutant library to bioconvert several flavonoids, BphA1 (1-22; T324A and I325L) and BphA1 (2-2; T324L and I325I) were selected. When expressed in E. coli together with bphA2A3A4B from strain KF707, both BphA1 (1-22) and BphA1 (2-2) bioconverted the refractory flavonoids, 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin), which were hardly converted by any unmodified and artificially-modified shuffled biphenyl dioxygeneses, into their vicinal diol forms, i.e., 2-(2,3-dihydroxyphenyl)-7-hydroxy-chromen-4-one and 2-(2,3-dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one, respectively. In addition, trans-chalcone was converted into 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one and further into 1,3-bis-(2,3-dihydroxyphenyl)-propan-1-one. The antioxidative activity of these generated compounds was markedly higher than that of the original substrates used.
Kagami
Osamu
O
Marine Biotechnology Institute, Kamaishi, Iwate, Japan.
Shindo
Kazutoshi
K
Kyojima
Akiko
A
Takeda
Kazuyo
K
Ikenaga
Hiroshi
H
Furukawa
Kensuke
K
Misawa
Norihiko
N
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001711:Biotransformation; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D005419:Flavonoids / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D016296:Mutagenesis; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology / Q000254:growth & development; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
D017931:DNA Primers; D005419:Flavonoids; C043561:chrysin; D010105:Oxygenases; C092087:biphenyl-1,2-dioxygenase; C429845:7-hydroxyflavone