Anaerobic metabolism of phenol in proteobacteria and further studies of phenylphosphate carboxylase
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ORIGINAL PAPER
Anaerobic metabolism of phenol in proteobacteria and further studies of phenylphosphate carboxylase Sirko Schmeling • Georg Fuchs
Received: 14 August 2009 / Revised: 1 October 2009 / Accepted: 1 October 2009 / Published online: 17 October 2009 Ó Springer-Verlag 2009
Abstract Anaerobic phenol metabolism was studied in three facultative aerobic denitrifying bacteria, Thauera aromatica, ‘‘Aromatoleum aromaticum’’ strain EbN1 (Betaproteobacteria), and Magnetospirillum sp. (Alphaproteobacterium). All species formed phenylphosphate and contained phenylphosphate carboxylase but not phenol carboxylase activity. This is in contrast to direct phenol carboxylation by fermenting bacteria. Antisera raised against subunits of the Thauera phenylphosphate synthase and phenylphosphate carboxylase partly cross-reacted with the corresponding proteins in the other species. Some unsolved features of phenylphosphate carboxylase were addressed in T. aromatica. The core sub-complex of this enzyme consists of three different subunits and catalyzes the exchange of 14CO2 with the carboxyl group of 4-hydroxybenzoate, but not phenylphosphate carboxylation. It was inactivated by oxygen or by the oxidizing agent thionin and fully reactivated under reducing conditions. The purified recombinant phosphatase subunit alone had only low phenylphosphate phosphatase activity in the absence of the other components. However, activity was strongly enhanced in the presence of the core enzyme resulting in phenylphosphate carboxylation. Hence, a tight interaction of the carboxylase subunits is required for dephosphorylation of phenylphosphate, which is coupled to the concomitant carboxylation of the produced phenolate to 4-hydroxybenzoate, thus preventing a futile cycle.
Communicated by Wolfgang Buckel. S. Schmeling G. Fuchs (&) Mikrobiologie, Fakulta¨t fu¨r Biologie, Universita¨t Freiburg, Scha¨nzlestr. 1, 79104 Freiburg, Germany e-mail: [email protected]
Keywords Anaerobic metabolism of phenol Phenylphosphate synthase Phenylphosphate carboxylase Thauera aromatica Aromatoleum aromaticum strain EbN1 Magnetospirillum sp. strain CC-26
Introduction Phenolic compounds are widely distributed in nature. More than 4,000 phenolic plant compounds are known, e.g. lignin and its precursors, tannins, flavonoids, quinones, and aromatic amino acids. Next to carbohydrates, they constitute the second most abundant class of organic compounds and thus important substrates for microbial growth. The aerobic metabolism of phenol has been studied in detail (Harwood and Parales 1996; Hayaishi 1994; Solyanikova and Golovleva 1999) and requires molecular oxygen as cosubstrate. Monooxygenases initiate the degradation of phenol by hydroxylation to catechol, one of the few central intermediates of aromatic metabolism. This is followed by an oxygen-dependent ortho or meta ring cleavage, which is accomplished by ring cleaving dioxygenases. Anaerobic metabolism of phenol, which has been reported for various pure cultures of strict anaerobic
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