1.14.13.44: 2-hydroxybiphenyl 3-monooxygenase

the variant exhibits 7% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl, suggesting inhibition of substrate entrance into the active site by the large aromatic residue

I244V

mutant enzyme has a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and 2-hydroxybiphenyl. The Km-value for guaiacol decreases with this mutant, but the Km-value for 2-hydroxybiphenyl increase

438847

M223A

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

M223E

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

M223I

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

M223K

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

M223Q

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

M321A

site-directed saturation mutagenesis, the mutant variant demonstrates altered regioselectivity by oxidizing 3-hydroxybiphenyl, and thus enabling the production of a distinct antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity to the well-studied piceatannol. Mutant enzyme crystal structure analysis and comparison to the wild-type enzyme structure

M321F

site-directed saturation mutagenesis, wild-type HbpA possess pro-S enantioselectivity towards the production of several chiral sulfoxides, whereas the mutant M321F exhibits improved enantioselectivity and increased activity compared to wild-type

M321L

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

M321V

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

P320X

site-directed mutagenesis, all mutations of Pro320 in the library result in activity loss. Therefore, it appears that a proline at this position is crucial for the catalytic reaction. The short distance of Pro320 from the FAD cofactor implies its involvement in enabling the movement of FAD, which is imperative for HbpA activity. Pro320 is conserved among flavin monooxygenases. In aklavinone-11-hydroxylase (RdmE), para-hydroxybenzoate hydroxylase (pHBH), and phenol hydroxylase (PH), this position next to the FAD is occupied by Pro315, Pro293, and Pro364, respectively

R242A

the variant is not active on NADH and 2,3-dihydroxybiphenyl

R242E

the variant is not active on NADH and 2,3-dihydroxybiphenyl

R242Q

the variant is not active on NADH and 2,3-dihydroxybiphenyl

V368A/L417F

double replacement improves the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the Vmax is increased and the Km-value is decreased. With 2-tert-butylphenol as substrate the turnover number is increased more than 5fold

438847

W225A

the variant exhibits 7% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl

W225Y

the variant exhibits 122% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl

W97A

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme

W97Y

site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme