p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

Abstract

A bacterium, strain PC-07, previously isolated as part of a coculture capable of growing on p-cresol under anaerobic conditions with nitrate as the acceptor was identified as an Achromobacter sp. The first enzyme of the pathway, p-cresol methylhydroxylase, which converts its substrate into p-hydroxybenzyl alcohol, was purified. The enzyme had an Mr of 130,000 and the spectrum of a flavocytochrome. It was composed of flavoprotein subunits of Mr 54,000 and cytochrome subunits of Mr 12,500. The midpoint redox potential of the cytochrome was 232 mV. The Km and kcat for p-cresol were 21 microM and 112 s-1 respectively, and the Km for phenazine methosulfate, the artificial acceptor used in the assays, was determined to be 1.7 mM. These properties place the enzyme in the same class as the p-cresol methylhydroxylases from aerobically isolated Pseudomonas spp.