2-Hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas azelaica is an NADH-dependent flavoprotein, that catalyzes the first step in the degradation pathway of 2-hydroxybiphenyl. To date, there is no crystal structure of the enzyme and the catalytic mechanism is unknown. This monooxygenase can serve as a biocatalyst for diphenol synthesis (e.g. hydroxytyrosol, a potent antioxidant), chiral sulfoxides (e.g. armodafinil, a wake-promoting drug), colorants (e.g. indole), and more. We have characterized the enzyme and determined its crystal structure at a resolution of 2.3Å. The entrance of the substrate to the active site was identified. Two residues, Ile198 and Gly255, were found to have an important role in the positioning of the substrate in the correct orientation. Substitution of Ile198 to Ala decreased enzyme affinity (Km) to the substrate by 75% with 25% improvement of the specific activity. However, replacement of Gly255 to Phe seems to block the entrance to the active site leading to a drastic decrease of 95% in activity. In addition, Trp225 and Arg242 were also found to be involved in the catalytic reaction. Variant W225A led to uncoupled NADH oxidation from product formation, while W225Y improved the activity by 130%. Variant R242E had decreased activity by 90%, while R242Q and R242A had no activity at all. The ability of HbpA to produce hydroxytyrosol from different tyrosol isomers was examined. HbpA had only low activity on o-tyrosol, while I244V improved its activity by 17%. The new insights on HbpA structure and function will be utilized for tailoring biocatalysts with improved activity and selectivity towards chiral sulfoxides.
