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

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; ␣ 2 ␤ 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 ␣ subunit of the enzyme was present in two isoforms, suggesting an ␣␣␤ 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.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…The deduced pcmUVWX gene products show similarities to proteins involved in the maturation of molybdenum-containing enzymes and are proposed to play a role in the molybdenum cofactor assembly of 4-hydroxybenzoyl-CoA reductase. On the other hand, the pcmQ gene may encode a MarR-type transcriptional regulator that could be involved in the expression of other pcm genes (277). Proteomic (277) and transcriptomic (48) results have shown that the pcmRST genes are induced not only when the G. metallireducens cells grow in the presence of 4-HBA but also when they are cultivated with benzoate.…”

“…7). Notably, although PcmR would be the FAD-containing subunit of 4-hydroxybenzoyl-CoA reductase, it does not contain the [4Fe-4S] cluster binding loop, which suggests a mode of electron transfer to the substrate that is different from that postulated for the classical 4-hydroxybenzoyl-CoA reductases from facultative anaerobes (277). The deduced pcmUVWX gene products show similarities to proteins involved in the maturation of molybdenum-containing enzymes and are proposed to play a role in the molybdenum cofactor assembly of 4-hydroxybenzoyl-CoA reductase.…”

“…Proteomic (277) and transcriptomic (48) results have shown that the pcmRST genes are induced not only when the G. metallireducens cells grow in the presence of 4-HBA but also when they are cultivated with benzoate. However, 4-hydroxybenzoyl-CoA reductase activity was detected in G. metallireducens cells grown in 4-HBA but not in cells grown in benzoate, suggesting a posttranslational regulation of this enzyme activity (277). An in silico search of the G. metallireducens genome indicated that bamY appears to be the only gene coding for a typical aromatic carboxylic acidCoA ligase (Fig.…”

“…The genome of S. aciditrophicus contains four different genes (syn1638, syn2417, syn2896, and syn2898) that are predicted to encode AMP-forming ligases that could activate aromatic or alicyclic compounds (237). Out of these four genes, syn1638 is the only one that is located in a gene cluster (Sa2) that also contains genes which have been shown to be involved in the reaction steps after benzoylCoA reduction (206,277,279), and therefore, it is assumed to be the bamY ortholog (bamY Syn ) in S. aciditrophicus (Fig. 2B).…”

“…The anaerobic catabolism of 4-HBA has also been studied for the obligate anaerobic bacterium G. metallireducens. Orthologs of the genes encoding 4-hydroxybenzoyl-CoA reductase from facultative anaerobes have been identified within the large genomic island of G. metallireducens (48,277). Thus, the pcmRST genes will encode the three-subunit 4-hydroxybenzoyl-CoA reductase from G. metallireducens (Fig.…”