P-Hydroxybenzoate hydroxylase

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... 

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... 

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. 

Ridder L, JN Harvey, IMCM Rietjens, J Vervoort, AJ Mulholland (2003) Ab initio QM/MM modeling of the hydroxylation step in p-hydroxybenzoate hydroxylase. J. Phys. Chem. B 107 (9) 2118-2126... 

Senn HM, Thiel S, Thiel H (2005) Enzymatic hydroxylation in p-hydroxybenzoate hydroxylase a case study for QM/MM molecular dynamics. J. Chem. Theory Comput. 1 494-505... 

Fig. J2. Chain trace of p-hydroxybenzoate hydroxylase. Stereo drawing from the work of Drenth and colleagues [60].

Mata, R. A. Werner, H.-J. Thiel, S. Thiel, W. Toward accnrate barriers for enzymatic reactions QM/MM case stndy on p-hydroxybenzoate hydroxylase, J. Chem. Phys 2008,128, 025104-025108. 

Westphal AH, Matorin A, Hink MA, Borst JW, van Berkel WJH, Visser AJWG. Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase. J. Biol. Chem. 2006 281 11074-11081. 

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