Genes, Enzymes, and Regulation of para -Cresol Metabolism in Geobacter metallireducens

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Abstract
In aerobic and facultatively anaerobic bacteria, the degradation of para -cresol ( p -cresol) involves the initial hydroxylation to p -hydroxybenzyl alcohol by water catalyzed by the soluble, periplasmatic flavocytochrome p -cresol methylhydroxylase (PCMH; α 2 β 2 composition). In denitrifying bacteria the further metabolism proceeds via oxidation to p -hydroxybenzoate, the formation of p -hydroxybenzoyl-coenzyme A (CoA), and the subsequent dehydroxylation of the latter to benzoyl-CoA by reduction. In contrast, the strictly anaerobic Desulfobacterium cetonicum degrades p -cresol by addition to fumarate, yielding p -hydroxybenzylsuccinate. In this work, in vitro enzyme activity measurements revealed that the obligately anaerobic Geobacter metallireducens uses the p -cresol degradation pathway of denitrifying bacteria. Surprisingly, PCMH, which is supposed to catalyze both p -cresol hydroxylation and p -hydroxybenzyl alcohol oxidation to the corresponding aldehyde, was located in the membrane fraction. The α subunit of the enzyme was present in two isoforms, suggesting an αα′β 2 composition. We propose that the unusual asymmetric architecture and the membrane association of PCMH might be important for alternative electron transfer routes to either cytochrome c (in the case of p -cresol oxidation) or to menaquinone (in the case of p -hydroxybenzyl alcohol oxidation). Unusual properties of further enzymes of p -cresol metabolism, p -hydroxybenzoate-CoA ligase, and p -hydroxybenzoyl-CoA reductase were identified and are discussed. A proteomic approach identified a gene cluster comprising most of the putative structural genes for enzymes involved in p -cresol metabolism ( pcm genes). Reverse transcription-PCR studies revealed a different regulation of transcription of pcm genes and the corresponding enzyme activities, suggesting the presence of posttranscriptional regulatory elements.