Identification and characterization of a chc gene cluster responsible for the aromatization pathway of cyclohexanecarboxylate degradation in Sinomonas cyclohexanicum ATCC 51369

Cyclohexanecarboxylate (CHCA) is formed by oxidative microbial degradation of n-alkylcycloparaffins and anaerobic benzoate, also known to be a synthetic intermediate or the starter unit biosynthesis cellular constituents secondary metabolites. Although two pathways have been proposed, genetic information has limited β-oxidation-like pathway. In this study, we identified gene cluster, designated chcC1XTC2B1B2RAaAbAc, that responsible for CHCA aromatization pathway in Sinomonas (formerly Corynebacterium) cyclohexanicum strain ATCC 51369. Reverse transcription-PCR analysis indicated chc cluster inducible it consists transcriptional units, chcC1XTC2B1B2R chcAaAbAc. Overexpression various genes Escherichia coli, purification recombinant proteins led functional characterization ChcAaAbAc as subunits cytochrome P450 system hydroxylation; ChcB1 ChcB2 trans-4-hydroxyCHCA cis-4-hydroxyCHCA dehydrogenases, respectively; ChcC1 was 4-oxoCHCA desaturase containing covalently bound FAD; ChcC2 4-oxocyclohexenecarboxylate desaturase. The binding constant ChcAa found 0.37 mM. Kinetic parameters established high catalytic efficiency towards compared trans- cis-4-hydroxyCHCA. Km Kcat values were 0.39 mM 44 s−1, respectively. Taken together with previous work on identification pobA encoding 4-hydroxybenzoate hydroxylase, now localized remaining set final protocatechuate before entry into tricarboxylic acid cycle.