Hydroxybenzoate hydroxylase

Gao X, CL Tan, CC Yeo, CL P (2005) Molecular and biochemical characterization of the x/ D-encoded 3-hydroxybenzoate 6 hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9S61. J Bacterial 187 7696-7702. 

Wong CM, MJ Dilworth, AR Glenn (1994) Cloning and sequencing show that 4-hydroxybenzoate hydroxylase Poba is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum. Microbiology (UK) 140 2775-2786. 

Flavoprotelns introducing oxygen at site adjacent to existing hydroxy group 4-hydroxybenzoate hydroxylase Anthranilate hydroxylase in yeasts Salicylate hydroxylase... 

Entsch B, DP Ballou (1989) Purification, properties, and oxygen reactivity of /)-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa. Biochim Biophys Acta 999 253-260. 

Howell JG, T Spector, V Massey (1972) Purification and properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. J Biol Chem 247 4340-4350. 

Van der Bolt FJT, van den Heuvel RHH, Vervoort J, van Berkel WJH (1997) 19F NMR study on the regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate by wild-type and Y385F p-hydroxybenzoate hydroxylase evidence for a consecutive oxygenolytic dehalogenation. Biochemistry 36 14192-14201... 

Cytochrome c peroxidase domain 1 C. Miscellaneous antiparallel a Carp muscle calcium-binding protein Egg lysozyme Citrate synthase Catalase domain 2 Cytochrome c peroxidase domain 2 p-Hydroxybenzoate hydroxylase domain 3 II. Parallel a/j3 domains... 

Aspartate transcarbamylase catalytic domain 1 Phosphofructokinase domain 1 p-Hydroxybenzoate hydroxylase domain 1 Glucosephosphate isomerase domain 1 Glutathione peroxidase C. Miscellaneous parallel a/j3 Carboxypeptidase Thioredoxin Carbonic anhydrase Phosphofructokinase domain 2 Glucosephosphate isomerase domain 2 III. Antiparallel /3 domains... 

Glyceraldehyde-phosphate dehydrogenase domain 2 Bacteriochlorophyll protein p-Hydroxybenzoate hydroxylase domain 2 Influenza virus hemagglutinin HA2 L7/L12 ribosomal protein... 

Glutathione reductase d2, d3 Influenza virus hemagglutinin HA1, HA2 p-Hydroxybenzoate hydroxylase (4-hydroxybenzoate 4-monooxygenose) dl, d2... 

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... 

In the active site of a hydroxylase, an OH group can be transferred from the peroxide to a suitable substrate (Eq. 18-42). Although radical mechanisms are likely to be involved, such hydroxylation reactions can also be viewed as transfer of OH+ to the substrate together with protonation on the inner oxygen atom of the original peroxide to give a 4a - OH adduct. The latter is a covalent hydrate which can be converted to the oxidized flavin by elimination of H20. This hydrate is believed to be the third spectral intermediate identified during the action of p-hydroxybenzoate hydroxylase 286 287 290... 

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... 

The enzyme p-hydroxybenzoate hydroxylase utilizes a cosubstrate together with 02 to form 3,4-dihydroxybenzoate. Indicate the mechanisms by which the bound FAD cofactor participates in the reaction. 

Figure 3. Reaction stoichiometries for p-hydroxybenzoate hydroxylase with substrate or coenzyme...

Preliminary studies in our laboratory with orcinol hydroxylase, from a pseudomonad, suggested that the 1-dFAD-reconstituted enzyme would catalyze NADH oxidation, but 02 was reduced exclusively to H202. No oxygen transfer yielding trihydroxytoluene could be detected. Because of instability of both trihydroxytoluene oxygenation product and orcinol hydroxylase apoenzyme, the 1-dFAD studies were pursued next in collaboration with Massey, Husain, Ballou, and Entsch at University of Michigan with the most well characterized bacterial flavoenzyme hydroxylase, p-hydroxybenzoate hydroxylase (2, 26, 27). The apoenzyme was reconstituted stably with 1-deazaFAD... 

Figure 4. Possible flavin peroxide and 1-deazaflavin peroxide species in p-hydroxybenzoate-hydroxylase-mediatea catalytic sequence...

However, recent x-ray studies on p-hydroxybenzoate-p-hydroxybenzoate hydroxylase binary complex crystals clearly show the aromatic substrate is bound at the flavin 4a-5 edge and orthogonal to the isoalloxazine plane (29). Unless this binary complex structure is highly misinformative, it can be inferred that in the 02, p-hydroxy-benzoate, enzyme ternary active complex, oxygen transfer is in the 4a,5 region, not the la, 1 region of the bound FAD, which rules out la-OOH derivatives as important oxygenating intermediates for this enzyme. 

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