טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBregman Cohen Almog
SubjectProtein Engineering of 2-Hydroxybiphenyl 3-Monooxygenase
for Modulating Activity and Selectivity
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract

2-Hydroxybiphenyl 3-monooxygenase (HbpA) from Pseudomonas azelaica HBP1 is an FAD-dependent monooxygenase which catalyzes the ortho-hydroxylation of a broad range of 2-substituted phenols in the presence of NADH and molecular oxygen. The enzyme was previously studied for its substrate range and with different NADH recycling systems, yet in the absence of a crystal structure, the contribution of residues in the active site to its function and selectivity remained unknown.

The goal of this research was to crystalize HbpA, alter its regio- and enantiospecificity using protein engineering and to shed light on structure-function correlations. The new insights on HbpA structure and mechanism were applied to identify variants with the ability to synthesize antioxidants and chiral sulfoxides.

We have determined the structure of HbpA with bound 2-hydroxybiphenyl (2HBP), the natural substrate, as well as several variants, at a resolution of 2.3-2.8 Å. An observed hydrogen bond between 2HBP and His48 in the active site confirmed the previously suggested role of this residue in substrate deprotonation. In order to modulate HbpA activity and selectivity, several residues in the active site were investigated by both specific and saturation mutagenesis. Residues Arg242 and Pro320 are suggested to facilitate FAD movement. It is proposed that Trp225 facilitates proper substrate entrance into the binding pocket, while Trp97 stabilizes the substrate in the active site. Met223 and D222 are involved in NADH entrance or binding to the active site, while M321 affects electron transfer from NADH to the FAD cofactor.

HbpA was found to possess mild pro-S enantioselectivity towards the production of several chiral sulfoxides, while variant M321F exhibited improved enantioselectivity. Variant M321A demonstrated altered regioselectivity by oxidizing for the first time 3-hydroxybiphenyl, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity as the well-studied piceatannol. The crystal structure of M321A was determined and molecular docking of the 3-substituted phenol provided a rational explanation for the altered regioselectivity.

A new NADH recycling system was successfully coupled to HbpA in vitro. The system is based on a soluble hydrogenase bound to carbon particles, termed the C@Enzyme. The benefit of this system is the use of H2 as an electron donor and thus it has 100% atom efficiency and no by-products.

Furthermore, to treat wastewater contamination by 2HBP, an antifungal agent used in post harvest treatment of fruits, we developed a biocatalyst expressing the enzymes HbpC and HbpD, which can be further combined with HbpA activity. In E. coli, the enzymes are highly expressed and could be manipulated by protein engineering methods. The feasibility of this system was illustrated with purified enzymes and in whole cells, presenting more than 80% conversion, resulting in benzoic acid formation.

In summary, protein engineering was used to study structure-function correlations of HbpA and to alter HbpA regioselectivity towards the production of a new antioxidant. In addition, first evidence of the potential of HbpA as a biocatalyst for chiral sulfoxides production was illustrated. Additionally, development of a degradation pathway for the pollutant 2HBP was proposed, coupling HbpA activity with HbpC and HbpD.