Publications

• Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. strain SE19 by in vitro transposition.

  Cheng Q., Thomas S.M., Kostichka K., Valentine J.R., Nagarajan V.

  Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as ... >> More

  Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway. << Less

  J. Bacteriol. 182:4744-4751(2000) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Cloning and characterization of four enzymes responsible for cyclohexylamine degradation from Paenarthrobacter sp. TYUT067.

  Feng K., Qi N., Jin Q., Gao L., Zhang J., Tian Q.

  Paenarthrobacter sp. TYUT067 is a soil bacterium that can degrade and use cyclohexylamine as the sole source of carbon and energy. However, the responsible enzymes involved in cyclohexylamine degradation by TYUT067 have not been cloned and characterized in detail yet. In this study, four possible ... >> More

  Paenarthrobacter sp. TYUT067 is a soil bacterium that can degrade and use cyclohexylamine as the sole source of carbon and energy. However, the responsible enzymes involved in cyclohexylamine degradation by TYUT067 have not been cloned and characterized in detail yet. In this study, four possible cyclohexylamine degradation genes, one cyclohexylamine oxidase (Pachao), two cyclohexanone monooxygenases (Pachms) and one lactone hydrolase (Pamlh) were successfully cloned and heterologous expressed in Escherichia coli T7 host cells. The four enzymes were purified and characterized. The optimal pH and temperature of the purified enzymes toward their own substrates were 7.0 (PaCHAO), 8.0 (PaCHM1), 9.0 (PaCHM2 and PaMLH) and 30 °C (PaCHAO and PaMLH), 40 °C (PaCHM2) and 45 °C (PaCHM1), respectively, with K_M of 1.1 mM (PaCHAO), 0.1 mM (PaCHM1), 0.1 mM (PaCHM2) and 0.8 mM (PaMLH), and yielding a catalytic efficiency k_cat/K_M of 16.1 mM^-1 s^-1 (PaCHAO), 1.0 mM^-1 s^-1 (PaCHM1), 5.0 mM^-1 s^-1 (PaCHM2) and 124.4 mM^-1 s^-1 (PaMLH). In vitro mimicking the cyclohexylamine degradation pathway was conducted by using the combined three cyclohexylamine degradation enzymes (PaCHAO, PaCHM2 and PaMLH) with 10-50 mM cyclohexylamine, 100% conversion of cyclohexylamine could be finished within 12 h without any detected intermediates. The current study confirmed the enzymes responsible for cyclohexylamine degradation in TYUT067 for the first time, provide basic information for further investigation and application of these specific enzymes in pollution control. << Less

  Protein Expr Purif 198:106136-106136(2022) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis.

  Sheng D., Ballou D.P., Massey V.

  Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon-caprolactone. The reaction catalyzed involves the four-electron reduction of O2 at the expense of a two-electron oxidation of NADP ... >> More

  Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon-caprolactone. The reaction catalyzed involves the four-electron reduction of O2 at the expense of a two-electron oxidation of NADPH and a two-electron oxidation of cyclohexanone to form epsilon-caprolactone. Previous studies suggested the participation of either a flavin C4a-hydroperoxide or a flavin C4a-peroxide intermediate during the enzymatic catalysis [Ryerson, C. C., Ballou, D. P., and Walsh, C. (1982) Biochemistry 21, 2644-2655]. However, there was no kinetic or spectral evidence to distinguish between these two possibilities. In the present work we used double-mixing stopped-flow techniques to show that the C4a-flavin-oxygen adduct, which is formed rapidly from the reaction of oxygen with reduced enzyme in the presence of NADP, can exist in two states. When the reaction is carried out at pH 7.2, the first intermediate is a flavin C4a-peroxide with maximum absorbance at 366 nm; this intermediate becomes protonated at about 3 s(-1) to form what is believed to be the flavin C4a-hydroperoxide with maximum absorbance at 383 nm. These two intermediates can be interconverted by altering the pH, with a pK(a) of 8.4. Thus, at pH 9.0 the flavin C4a-peroxide persists mainly in the deprotonated form. Further kinetic studies also demonstrated that only the flavin C4a-peroxide intermediate could oxygenate the substrate, cyclohexanone. The requirement in catalysis of the deprotonated flavin C4a-peroxide, a nucleophile, is consistent with a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone. In the course of these studies, the Kd for cyclohexanone to the C4a-peroxyflavin form of CHMO was determined to be approximately 1 microM. The rate-determining step in catalysis was shown to be the release of NADP from the oxidized enzyme. << Less

  Biochemistry 40:11156-11167(2001) [PubMed] [EuropePMC]