4- HYDROXYBENZOATE 3-MONOOXYGENASE

The 4-methoxybenzoate monooxygenase from Pseudomonas putida shows low substrate specificity. Although it introduces only a single atom of oxygen into 3-hydroxy- and 4-hydroxybenzoate, it accomplishes the conversion of 4-vinylbenzoate into the corresponding side-chain diol (Wende et al. 1989). 

Eppink MHM, SA Boeren, J Vervoort, WJH van Berkel (1997) Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604. J Bacterial 179 668-6687. 

Oxidative decarboxylation of hydroxybenzoates by the yeast Candida parapsilosis is catalyzed by a flavin monooxygenase that is able to use a range of fluorinated hydroxybenzoates that were examined by F NMR (Eppink et al. 1997). 

The monooxygenase from Pseudomonasfluorescens that converts 4-hydroxybenzoate into 3,4-dihydroxybenzoate before ring hssion has been characterized (Howell et al. 1972). 

The following is review on the molecular and physical properties of this class of monooxygenases, which are also known as hydroxylases. A typical monooxygenase reaction is the hydroxylation of an alkane to an alcohol which involves a reduced cosubstrate that reduces a second atom within the O2 molecule to form water. Flavin-containing monooxygenases include lysine oxygenase and 4-hydroxybenzoate hydroxylase. Reduced pteri-dines are involved in the phenylalanine hydroxylase and tryptophan hydroxylase reactions. See also Cytochrome P-450... 

Tire tetrahydrobiopterin formed in this reaction is similar in structure to a reduced flavin. The mechanism of its interaction with 02 could reasonably be the same as that of 4-hydroxybenzoate hydroxylase. However, phenylalanine hydroxylase, which catalyzes the formation of tyrosine (Eq. 18-45), a dimer of 451-residue subunits, contains one Fe per subunit,113 313i whereas flavin monooxygenases are devoid of iron. Tyrosine hydroxylase416 193 and tryptophan hydroxylase420 have very similar properties. All three enzymes contain regulatory, catalytic, and tetramerization domains as well as a common Fe-binding motif in their active sites.413 421 4213... 

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... 

Another example of a monooxygenase, jo-hydroxybenzoate hydroxylase (HBH), makes use of a flavin cofactor in combination with a reductase (equation 127). ... 

Thus, these flavin-dependent monooxygenases demonstrate the capacity of enzymes to modify the pK of reactants through specific interactions of amino acid residue side-chains with the substrate. In principle, the catalytic process depicted in Fig. 4.81 is impossible outside the active site of the enzyme, since in solution at any given pH the simultaneous generation of both the protonated C(4a)-hydroperoxyflavin and the deprotonated para-hydroxybenzoate substrate would be impossible due to the pK values of these two reactants. The enzyme provides a way for simultaneous generation of the activated protonated C(4a)-hydroperoxyflavin cofactor and the activated deprotonated substrate. 

Bacterial monooxygenases that are flavoproteins requiring NADPH are involved in the hydroxylation of a number of phenolic compounds (a) phenol and chlorophenols, (b) salicylate, (c) 4-hydroxybenzoate, (d) 4-hydroxyphenylacetate, and (e) anthranilate in fungi. Further details of some of these are given later in this chapter and in Chapter 6 (Sections 6.2.1 and 6.5.1.2). 

The mechanism for the hydroxylation of aromatic substrates by flavoprotein monooxygenases has been the subject of signiflcant research interest and controversy over the past decade. These enzymes (p-hydroxybenzoate hydroxylase, phenol hydroxylase, and melilotate hydroxylase) catalyze the initial step in the )8-ketoadipic acid pathway, the hydroxylation of substituted phenols into catechols (Scheme 55). Oxygen is required as cosubstrate, which is activated by the reduced FAD cofactor. The complex mechanism for the oxidative half-reaction is thought to consist of at least four steps and three intermediates 239-242) and to involve a controversial 4a,5-ring-opened flavin 242, 249, 250) (Scheme 56). The flavin C4a-hydroperoxy intermediate 64 and flavin C4a-hydroxy intermediate 65 have been assigned the structures shown in Scheme 56 based on the UV absorbance spectra of various model compounds compared with that of the modified enzyme cofactor alkylated at N(5) 243). However, evidence for the intermediacy of various ring-opened flavin species has been tentative at best, as model compounds and model reactions do not support such an intermediate 242). 

NADPH can serve as a cosubstrate of flavoprotein monooxygenase by first reducing the flavin, after which the reduced flavin can react with O2 to generate the hydroxylating reagent. An example is the bacterial 4-hydroxybenzoate hydroxylase which forms... 

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