Phenol Hydroxylase from Yeast

An enzyme, able to carry out an NADPH‐dependent hydroxylation of monocyclic phenols, was purified 20–30‐fold from Trichosporon cutaneum grown on phenol or resorcinol as a major carbon source. The purified enzyme was homogeneous upon analytical disc electrophoresis. The enzyme is a bright‐yellow protein with an absorption spectrum typical of flavoproteins. Its molecular weight is 148000, and its estimated FAD content is approximately 1 mole per mole enzyme. The purified enzyme has essentially the same broad substrate specificity as crude preparations. In addition to phenol it also hydroxylates the three isomeric diphenols and a number of phenol derivatives to their corresponding o ‐diols. Km values for the three substrates of the holoenzyme are all of the order 10 μM : K m (phenol) = 18 μM, K m (NADPH) = 71 μM. The absorption spectrum of the holoenzyme is modified in the presence of phenol. FAD could be resolved from the purified enzyme by preparative disc electrophoresis. Reassembly of the holoenzyme required SH‐groups. The enzyme activity is unaffected by chelators of iron and copper but it is inhibited by heavy metals. The inhibition by p ‐chloromercuribenzoate is readily reversed by dithiothreitol. Among oxidizing agents, hydrogen peroxide and peroxidase depressed the enzyme activity, whereas catalase was without effect. Among reducing agents ascorbate depressed enzyme activity. Sodium dithionite and sodium borohydride bleached the enzyme with concomitant loss of activity. After reduction with dithionite, the enzyme was rapidly re‐oxidized, re‐gaining its original activity. After reduction with borohydride re‐oxidation was very slow. However, the enzyme could be re‐activated by incubation with FAD. The enzyme is very sensitive to inorganic salts, nitrogen bases and detergents. Chloride is quite deleterious whereas phosphate seems to stabilize the enzyme.