Molecular ecology and biochemistry of cyclohexenoyl-CoA hydrolysing/forming enzymes

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In anaerobic bacteria aromatic hydrocarbons such as BTEX are degraded via the central intermediate benzoyl-CoA. The latter is further metabolized by enzymes of the so-called benzoyl-CoA degradation pathway present in all aromatic compound degrading anaerobic bacteria. In the preceding funding period we developed and applied a combination of functional PCR-based assays specific for conserved genes coding for characteristic enzymes of the benzoyl-CoA degradation pathway. The enzymes targeted comprised the ring cleaving hydrolase (bamA-assay), the benzene ring dearomatizing benzoyl-CoA reductases (BCRs) of facultative anaerobes (bcrC/bzdN-assays) and of obligate anaerobes (bamB-assay). The application of these assays at environmental sites was approved. In the next funding period we will apply/modify the assays for mRNA detection and extended applications (microarrays and/or amplicon sequencing). In a second subporoject we studied enzymes involved in a reverse benzoyl-CoA degradation pathway operating in Syntrophus aciditrophicus during crotonate and/or benzoate fermentation. Unexpectedly, we identified that a series of dienoyl-CoA/monoenoyl-CoA disproportionation reactions are involved in cyclohexanoyl-CoA formation. The enzymes involved in these unusual reactions will be isolated and characterized in the next funding period.