Publications

• Phenol hydroxylase from yeast. Sulfhydryl groups in phenol hydroxylase from Trichosporon cutaneum.

  Neujahr H.Y., Gaal A.

  Thiol groups in phenol hydroxylase were measured using two different -SH reagents and amino acid analysis. Stepwise blocking of the -SH groups was correlated with enzyme activity and FAD content. The results indicate that the enzyme contains 16 -SH groups per molecule of Mr 1.48 X 10(5). At least ... >> More

  Thiol groups in phenol hydroxylase were measured using two different -SH reagents and amino acid analysis. Stepwise blocking of the -SH groups was correlated with enzyme activity and FAD content. The results indicate that the enzyme contains 16 -SH groups per molecule of Mr 1.48 X 10(5). At least four -SH groups are not accessible without the use of a denaturing agent. There is seemingly no disulphide bridge. On the whole, the reactivity towards p-hydroxymercuribenzoate is much greater than towards 5,5'-dithio-bis(2-nitrobenzoic acid). The two reagents seem to have a different specificity with respect to which -SH groups they attack. Either reagent dislocates FAD from the holoenzyme, leaving a characteristic mercaptide derivative of the apoenzyme. Such derivatives were used to prepare the apoenzyme. The -SH groups in the apoenzyme are much more reactive towards 5,5'-dithio-bis(2-nitrobenzoic acid) than the -SH groups in the holoenzyme. The stoichiometry of the reaction with 5,5'-dithio-bis(2-nitrobenzoic acid) indicates that at least 8 -SH groups are located in spatially close pairs. The most reactive pair of all does not appear to be of importance for enzyme activity. The two subsequent -SH pairs are essential for enzyme activity are are involved in FAD attachment. The reactivity of the -SH groups decreases dramatically in the presence of substrate, even at substrate concentrations equivalent to the level of the catalytic sites. The isolated apoenzyme has a tendency to aggregate. A large proportion of -SH groups in such aggregate(s) is buried, especially when EDTA is not used throughout the preparation of the apoenzyme. The aggregates are enzymically inactive. << Less

  Eur J Biochem 58:351-357(1975) [PubMed] [EuropePMC]

• Phenol hydroxylase from yeast. Purification and properties of the enzyme from Trichosporon cutaneum.

  Neujahr H.Y., Gaal A.

  Eur. J. Biochem. 35:386-400(1973) [PubMed] [EuropePMC]

• The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis.

  Enroth C., Neujahr H.Y., Schneider G., Lindqvist Y.

  <h4>Background</h4>The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use fla ... >> More

  <h4>Background</h4>The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols.<h4>Results</h4>The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction.<h4>Conclusions</h4>Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis. << Less

  Structure 6:605-617(1998) [PubMed] [EuropePMC]