Pseudomonas putida N.C.I.B. 9869, when grown on 3,5-xylenol, hydroxylates the methyl groups on 3,5-xylenol and on p-cresol by two different enzymes. 3,5-Xylenol methylhydroxylase, studied only in relatively crude extracts, requires NADH, is not active with p-cresol and is inhibited by cyanide, but not by CO. The p-cresol methylhydroxylase requires an electron acceptor and will act under anaerobic conditions. It was purified and is a flavocytochrome c of mol.wt. approx. 114,000 consisting of two subunits of equal size. The enzyme catalyses the hydroxylation of p-cresol (Km 16 micron) and the further oxidation of product, p-hydroxybenzyl alcohol (Km 27 micron) to p-hydroxybenzaldehyde. A different p-cresol methylhydroxylase of the flavocytochrome c type is induced by growth on p-cresol. It too was purified and has mol.wt. approx. 100,000, and again consisted of two equal-size subunits. The Km for p=cresol 3.6 micron and for p=hydroxybenzyl alcohol, 15 micron.

Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.

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