Para-hydroxybenzoate hydroxylase

Modelling can pinpoint functional groups and analyse catalytic interactions. In several enzymes, catalytic interactions have been identified via calculation. For example, in the flavin-dependent monooxygenases, para-hydroxybenzoate hydroxylase and phenol hydroxylase, a conserved proline residue was found from QM/MM modelling, which specifically stabilizes the transition state for aromatic hydroxy-lation.12,13... 

Moran GR, Entsch B, Palfey BA, Ballou DP. Electrostatic effects on substrate activation in para-hydroxybenzoate hydroxylase studies of the mutant Lysine 297 Methionine. Biochemistry 1997 36 7548-7556. 

A recent study of para-hydroxybenzoate hydroxylase (PHBH) by Bidder et al. [49,50] provides an interesting example of the validation of QM/MM calculations on the enzyme mechanism by comparison with experimental data. The correlation found between calculated activation barriers and the logarithm of experimental rate constants for a series of alternative substrates also provides support for the proposed mechanism hydroxylation of hydroxylation. These studies are a good example of QM/MM reaction pathway calculations for an enzyme, including technical aspects of system set-up and practical considerations, and so will be outlined here in some detail. 

However, the activated, protonated C(4a)-hydroperoxyflavin is not by itself a powerful oxidizing species. From detailed biochemical studies on the catalytic mechanism of such enzymes, especially para-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PH), it appears that not only the peroxyflavin needs to be activated through protonation of the distal oxygen, but also the phenolic substrates require activation in order to obtain substrate conversion. This substrate activation is achieved through deprotonation of the hydroxyl moiety of the phenolic substrate. The active site of PHBH, for example, shows a tyrosine network consisting of tyrosines 385 and 201 (Fig. 4.82) responsible for this deprotonation and activation of the substrate. 

Fig. 4.82. Activation of para-hydroxybenzoate in the active site of para-hydroxybenzoate hydroxylase through deprotonation of its hydroxyl moiety by tyrosine residues 201 and 385.

Much of the work into the structure and mechanism of aromatic hydroxylation by Class A FPMOs has come from studies of para-hydroxybenzoate hydroxylase (PHBH), the structure of which was first determined in 1979 [44]. PHBH catalyses not only the hydroxylation of 4-hydroxybenzoate 24 but also 2,4-dihydroxybenzoate 26, 4-mercaptobenzoate 27, and fluorinated and chlorinated derivatives of the parent substrate (Figure 8.10). 

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. 

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