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

环己烷羧酸盐(CHCA) 由正烷基环烷烃的氧化微生物降解和苯甲酸盐的厌氧降解形成，也已知是合成中间体或细胞成分和次生代谢物生物合成的起始单元。尽管已经提出了两种降解途径，但遗传信息仅限于β-氧化样途径。在这项研究中，我们鉴定的基因簇，指定chcC1XTC2B1B2RAaAbAc，即负责在CHCA芳构化途径Sinomonas（原棒杆菌）C yclohexanicum菌株ATCC 51369.逆转录PCR分析表明，该CHCCHCA 可诱导基因簇，它由两个转录单位chcC1XTC2B1B2R和chcAaAbAc 组成。大肠杆菌中各种基因的过表达和重组蛋白的纯化导致 ChcAaAbAc 作为细胞色素 P450 系统亚基的功能表征，负责 CHCA 羟基化；ChcB1 和 ChcB2 作为反式-4-羟基CHCA和顺式-4-羟基CHCA脱氢酶，分别；ChcC1 被鉴定为含有共价结合的 FAD 的 4-oxoCHCA 去饱和酶；ChcC2 被鉴定为 4-氧代环己烯羧酸去饱和酶。发现 ChcAa 对 CHCA 的结合常数为 0.37 mM。为 ChcB1 建立的动力学参数表明，与反式或顺式-4-羟基CHCA 相比，它对 4-oxoCHCA 具有较高的催化效率。ChcC1 对 4-oxoCHCA的K _m和K _cat值分别为 0.39 mM 和 44 s ^-1。结合先前关于识别pobA 的工作 对于编码 4-羟基苯甲酸羟化酶的基因，我们现在已经定位了剩余的一组基因，用于在进入三羧酸循环之前最终降解原儿茶酸。