Hydroxybiphenyl monooxygenase

Scheme 3.19 Metalloenzyme ATHase S112A combined in a chemo-enzymatic cofactor recycling system for the oxygenation reaction catalyzed by the hydroxybiphenyl monooxygenase from P. azaleica.

The structure of PHBH is shown in Figure 8.12. It is an enzyme of 394 amino acids, with a three domain structure composed of an N-terminal "Rossman fold" domain that binds the ADP of the FAD coenzyme, a second domain that binds the para-hydroxybenzoate substrate and, a third, largely helical domain that is involved in dimerization wdth another subunit. The tricyclic isoalloxa-zine ring of the flavin is bound at the interface between the first two major domains, and there is known to be movement of flavin associated with the accommodation of substrate [46]. The structure of PHBH in the presence of NADPH has not been determined, but it is thought that there is only a transient association of the two cofactors to effect flavin reduction after which the NADP+ dissociates to permit substrate binding [46]. Although PHBH itself has not been the focus of studies in applications in synthesis, other Class A FPMO aromatic hydroxylases, such as hydroxybiphenyl monooxygenase from Pseudomonas azelaica have been used for the preparation of hydroxylated compounds. 

Reactions catalyzed by hydroxybiphenyl monooxygenase (HbpA) from P. azelaica. 

Hydroxybiphenyl Monooxygenase (HbpA) from Pseudomomas azelaica... 

The degradation of 2-hydroxybiphenyl by Pseudomonas azelaica HBPl is initiated by 2-hydroxybiphenyl 3-monooxygenase (Suske et al. 1999). 

Suske, W. A., M. Held, A. Schmidt, T. Fleischmann, M. G. Wubbolts, and H.-P. E. Kohler, Purification and characterization of 2-hydroxybiphenyl 3-monooxygenase, a novel NADH-dependent, FAD-containing aromatic hydroxylase from Pseudomonas azelaica HBP1 , J. Biol. Chem., 272, 24257-24265 (1997). 

In another system, the [Cp Rhm(bpy)(H20)]2+ complex was used for the regeneration of NADH in combination with an isolated flavine-dependent monooxygenase (2-hydroxybiphenyl-3-monooxygenase) from P. azelaica [120]. This enzyme catalyzes the specific ort/m-hydroxylation of several a-substituted phenol derivatives with simultaneous oxidation of NADH (Fig. 25). 

Fig. 25 Specific ortho-hydroxylation of a-substituted phenols catalyzed by NAD+-dependent 2-hydroxybiphenyl-3 -monooxygenase (HbpA).

Figure 16.3-8. Reaction scheme for the ortho-hydroxylation of phenol derivatives catalyzed by 2-hydroxybiphenyl-3-monooxygenase (HbpA).

J. Lutz, B. Krummenacher, B. Witholt, A. Schmid. 2-Hydroxybiphenyl 3-Monooxygenase Large Scale Preparation and Cell Free... 

The oxidation of phenols to catechols or hydroquinones by tyrosinase enzymes has been developed for biocatalysis. For example, the ortho-hydroxylation of L-tyrosine 162 (and also substituted variants) to give l-DOPA 163 has been extensively studied due to the importance of l-DOPA in the treatment of Parkinson s disease [92, 93]. An arene hydroxy lating enzyme having a broad substrate scope is 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica, which is able to oxidize many ortho-substituted phenols 68 to the corresponding catechols 127 [94], as shown in Scheme 32.19. A notable example of an industrial biocatalytic arene hydroxylation that has been employed on very large scale (lOOm fermentation) is the pora-hydroxylation of R)-2-phenoxypropionic acid 164 by whole cells of Beauveria bassiana Lu 700 to give (R)-2-(4-hydroxyphenoxy)propionic acid 165, an important intermediate in herbicide manufacture [95]. 

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