¹⁹F‐NMR study on the pH‐dependent regioselectivity and rate of the ortho‐hydroxylation of 3‐fluorophenol by phenol hydroxylase from Trichosporon cutaneum

Abstract

The regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon curuneum (EC 1.14.13.7) was studied using 19F-NMR. The regioselective hydroxylation as well as the rate of orrho-hydroxylation are pH dependent with a pKa of 6.5. At pH values below 6.5, 3-fluorophenol preferentially becomes hydroxylated at the C6 ortho position, resulting in a maximum C6/C2 hydroxylation ratio of 6.7. Upon increasing the pH, the total rate of conversion increases. Also, the C2 ortho-hydroxylation increases relatively to the C6 orthohydroxylation and yields a minimum C6/C2 hydroxylation ratio of 2.2 at pH values above 7.5.Based on data from I9F-NMR binding studies and molecular orbital calculations, a hypothesis is put forward which explains the pH-dependent effects observed. A mechanism is proposed involving an active-site amino acid residue acting as a base in the reduced form of the protein. Deprotonation of this residue results in hydrogen bond formation with the hydroxyl moiety of the phenolic substrate, leading to (partial) deprotonation of the substrate. Molecular orbital calculations demonstrate that such a (partial) deprotonation increases (a) the overall reactivity of 3-fluorophenol for an electrophilic attack and (b) the reactivity of C2 relative to the C6 position. The hypothesis may explain the decrease in the C6/C2 hydroxylation ratio. Furthermore the increased amount of orthohydroxylated products formed with increasing pH can also be explained by this hypothesis.Phenol hydroxylase from Trichosporoa cutaneurn is one of the many flavin-dependent aromatic hydroxylases [l 1. The enzyme catalyses the conversion of phenol to 1 ,Zdihydroxybenzene (catechol). The monooxygenase phenol hydroxylase is a homodimer (molecular mass 152 kDa), each subunit containing a non-covalently bound FAD [2]. Unlike most bacterial flavin-dependent aromatic hydroxylases, eukaryotic phenol hydroxylase shows a rather broad substrate specificity. In addition to the parent substrate phenol, the enzyme also catalyses the hydroxylation of various substituted phenols such as o-, m-and p-fluorophenol, m-and p-chlorophenol, aminophenols, dihydroxybenzenes and cresol [3, 41.The catalytic mechanism of the enzyme is reported to proceed by formation of a C(4a)-hydroperoxyflavin intermediate, formed upon two-electron reduction of the flavin prosthetic group by NADPH and incorporation of an oxygen molecule [3, 51. The mechanism by which the C(4a)-hydroperoxyflavin intermediate converts the substrate to its hydroxylated form is supposed to proceed analogously to the mechanism for other aromatic hydroxylases, i.e. by an elec- trophilic attack of the peroxide function, or a hydroxyl radical derived from it, on the substrate [6-lo]. The nature in which the substrate reacts, however, is still a matter of debate. Despite the availability of the recently determined amino acid sequence [ll], it is not clear which amino acid residues participate in catalysis. Binding of phenol was reported to be favored at higher pH va...

NIH shift in flavin-dependent monooxygenation: Mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase

“…This is supported by linear free energy-type correlations, which indicate that the flavin C(4a) group behaves as an acid (5,6). Studies with fluorinated phenols (7) and theoretical calculations on the reactivity of the peroxide (8) also are consistent with this type of mechanism. However, experimental evidence allowing the unequivocal identification of a specific mechanistic variant for the monooxygenation step has not yet emerged.…”

Conversion of Phenol Derivatives to Hydroxylated Products by Phenol Hydroxylase from Trichosporon cutaneum

“…A less negative E(HOM0) can be expected to result in a stronger interaction between the substrate HOMO and the C(4a)-hydroperoxyflavin lowest unoccupied molecular or- Molecular orbital characteristics of phenol derivatives of importance for nucleophilic attack by the reactive electrons of the phenol on the C(4a)-hydroperoxyflavin intermediate, as calculated by The calculated HOMO characteristics of the various phenol derivatives are presented in Table 5. Values for both the protonated and the deprotonated forms of the phenol derivatives are presented, because results of a previous study demonstrated that the phenolic substrates are likely to become partially deprotonated upon binding to the active site of phenol hydroxylase [23].…”

Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases

“…After substrate hydroxylation, the C4a-hydroxyflavin is formed. [43] In the final stage of the reaction (Figure 2) [44,45] In contrast to PHBH, PHHY shows significant uncoupling of substrate hydroxylation, even with the physiological substrate. [44] A slow hydroxylation rate is the main cause of the unproductive formation of hydrogen peroxide.…”