Publications

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

  Yamamoto T., Hasegawa Y., Lau P.C.K., Iwaki H.

  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 ... >> More

  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. << Less

  J. Biosci. Bioeng. 132:621-629(2021) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Purification and properties of 4-hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum.

  Obata H., Uebayasi M., Kaneda T.

  4-Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous ... >> More

  4-Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous state by ammonium sulfate precipitation and chromatography on DEAE-650s, agarose-NAD and hydroxyapatite. The enzyme consisted of two identical subunits and had a native relative molecular mass of 53,600. There were two residues each of cysteine and tryptophan in the enzyme molecule. Oxo acid rather than hydroxy acid was routinely used as substrate for assay of the enzyme. The enzyme is highly specific for 4-oxocyclohexanecarboxylic acid: the carboxyl group is essential and the position of carbonyl group is important; neither the 2-oxo nor the 3-oxo homologue was used as substrate. A methyl substitution on the ring of 4-oxocyclohexanecarboxylate resulted in an almost complete loss of its activity. The reduction product was identified as trans-4-hydroxycyclohexanecarboxylic acid by gas-liquid chromatography and mass spectrometry. It was used as a substrate for the reverse reaction in the presence of NAD but not its cis-isomer. The enzyme was specific for the B-side (pro-S) hydrogen of NADH in the hydrogen transfer from NADH to 4-oxocyclohexanecarboxylate. The Km values for 4-oxocyclohexanecarboxylate and NADH in the reduction reaction at pH 6.8 were 0.50 mM and 0.28 mM, respectively, whereas those for trans-4-hydroxycyclohexanecarboxylate and NAD in the oxidation reaction at pH 8.8 were 0.51 mM and 0.23 mM, respectively. The equilibrium constant of the reaction was 1.79 x 10(-10) M. The enzyme was strongly inhibited by N-bromosuccinimide. << Less

  Eur. J. Biochem. 174:451-458(1988) [PubMed] [EuropePMC]