Anaerobic oxidation of p-cresol mediated by a partially purified methylhydroxylase from a denitrifying bacterium

Abstract

Anoxic cell extracts of a denitrifying bacterial isolate were shown to oxidize p-cresol to phydroxybenzoate. Oxidation of the substrate was independent of molecular oxygen and required nitrate as the natural terminal electron acceptor. Two enzyme activities were implicated in the pathway utilized by PC-07. A p-cresol methylhydroxylase mediated the oxidation ofp-cresol to p-hydroxybenzaldehyde, which was further oxidized to p-hydroxybenzoate by an NAD+-dependent dehydrogenase. The PC-07 methylhydroxylase was partially purified by anion-exchange chromatography. The protein appeared to be a multifunctional flavocytochrome, which first oxidized p-cresol to p-hydroxybenzyl alcohol, which was then oxidized to p-hydroxybenzaldehyde. The identity of the aldehyde was confirmed by mass spectroscopy. The PC-07 methylhydroxylase had a limited substrate range and required an alkyl-substituted phenolic ring with a hydroxyl group in the para position. From the available evidence, p-cresol, a naturally occurring phenol, exhibited the greatest affinity to the enzyme and therefore may be its natural substrate.A p-cresol (pCr)-utilizing, denitrifying bacterium, PC-07, has been previously isolated and described (4). The isolate is one member of a bacterial coculture comprising two denitrifying species which interdependently utilize pCr as the sole source of carbon for growth under anaerobic conditions (5). Initially, substrate oxidation to p-hydroxybenzoate (pOHB) is mediated by the PC-07 isolate and provides the ring fission substrate to the second member of the coculture; neither isolate is able to use the respective substrate of its partner for growth under anaerobic conditions. The PC-07 isolate, however, can utilize pCr as its sole source of carbon for growth under aerobic conditions. Studies with whole-cell suspensions of PC-07 have established the pathway for the anaerobic oxidation of pCr to pOHB via p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde (pHBZ) intermediates. Substrate oxidation depends on the stoichiometric reduction of N03-to N2.The studies presented here further characterized the anaerobic oxidation of pCr by cell extracts of the PC-07 isolate. In addition, these studies described the partial purification and characterization of a pCr methylhydroxylase enzyme (PCMH) which mediates the initial oxidation of pCr. MATERIALS AND METHODS

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

“…1. This confirms the suggestion of Bossert et al (2) from the oxidized-reduced difference spectrum of the partially purified enzyme that it contained heme and flavin moieties. The ratio of 0.9 of the peak at 275 nm to that at 416 nm for the reduced enzyme is slightly lower than that for the similar enzyme from P. putida NCIB 9869.…”

“…Partial purification of the enzyme suggested that it was of a type similar to that from the Pseudomonas sp., containing flavin and cytochrome, although this description was considered preliminary until spectra of the purified enzyme were obtained (2). This is now confirmed here by the complete purification of the enzyme and characterization of its subunit structure.…”

“…Purification proved more difficult than for the Pseudomonas enzymes, and extra steps had to be incorporated to give pure enzyme. However, the final preparation represented at 600-fold purification, a considerable improvement on the partially purified preparation reported previously (2).…”

“…cally, this organism is only able to oxidize p-cresol to 4-hydroxybenzoate), Bossert et al (2) showed that whether the organism was grown aerobically or anaerobically, the partially purified enzyme had the same elution profile during purification and an identical substrate range and Km. The bacteria were harvested by using an Alpha Laval LAB 102B continuous flow centrifuge, giving a very loose pellet which was stored at -20°C.…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Methyl groups are removed to leave hydroxyl substituents. The anaerobic degradation of many industrially produced phenolics, including phenol and p-cresol (4-methylphenol), converge at the level of 4-HBA, which is also a common growth substrate for different anaerobic bacteria (38). The peripheral pathway for the anaerobic degradation of 4-HBA involves two catabolic steps, i.e., its CoA-dependent activation and further reductive dehydroxylation to benzoyl-CoA ( Fig.…”