Publications

• Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23. Sequencing, gene expression, purification, and characterization.

  Oinuma K., Hashimoto Y., Konishi K., Goda M., Noguchi T., Higashibata H., Kobayashi M.

  Analysis of the nitrile hydratase gene cluster involved in nitrile metabolism of Pseudomonas chlororaphis B23 revealed that it contains one open reading frame encoding aldoxime dehydratase upstream of the amidase gene. The amino acid sequence deduced from this open reading frame shows similarity ( ... >> More

  Analysis of the nitrile hydratase gene cluster involved in nitrile metabolism of Pseudomonas chlororaphis B23 revealed that it contains one open reading frame encoding aldoxime dehydratase upstream of the amidase gene. The amino acid sequence deduced from this open reading frame shows similarity (32% identity) with that of Bacillus phenylacetaldoxime dehydratase (Kato, Y., Nakamura, K., Sakiyama, H., Mayhew, S. G., and Asano, Y. (2000) Biochemistry 39, 800-809). The gene product expressed in Escherichia coli catalyzed the dehydration of aldoxime into nitrile. The Pseudomonas aldoxime dehydratase (OxdA) was purified from the E. coli transformant and characterized. OxdA shows an absorption spectrum with a Soret peak that is characteristic of heme, demonstrating that it is a hemoprotein. For its activity, this enzyme required a reducing reagent, Na2S2O4, but did not require FMN, which is crucial for the Bacillus enzyme. The enzymatic reaction was found to be catalyzed when the heme iron of the enzyme was in the ferrous state. Calcium as well as iron was included in the enzyme. OxdA reduced by Na2S2O4 had a molecular mass of 76.2 kDa and consisted of two identical subunits. The kinetic parameters of OxdA indicated that aliphatic aldoximes are more effective substrates than aromatic aldoximes. A variety of spectral shifts in the absorption spectra of OxdA were observed upon the addition of each of various compounds (i.e. redox reagents and heme ligands). Moreover, the addition of the substrate to OxdA gave a peak that would be derived from the intermediate in the nitrile synthetic reaction. P. chlororaphis B23 grew and showed the OxdA activity when cultured in a medium containing aldoxime as the sole carbon and nitrogen source. Together with these findings, Western blotting analysis of the extracts using anti-OxdA antiserum revealed that OxdA is responsible for the metabolism of aldoxime in vivo in this strain. << Less

  J. Biol. Chem. 278:29600-29608(2003) [PubMed] [EuropePMC]

• Aldoxime dehydratase co-existing with nitrile hydratase and amidase in the iron-type nitrile hydratase-producer Rhodococcus sp. N-771.

  Kato Y., Yoshida S., Xie S.X., Asano Y.

  We identified an aldoxime dehydratase (Oxd) gene in the 5'-flanking region of the nitrile hydratase-amidase gene cluster in the photoreactive iron-type nitrile hydratase-producer, Rhodococcus sp. N-771. The enzyme showed 96.3%, 77.6%, and 30.4% identities with the Oxds of Rhodococcus globerulus A- ... >> More

  We identified an aldoxime dehydratase (Oxd) gene in the 5'-flanking region of the nitrile hydratase-amidase gene cluster in the photoreactive iron-type nitrile hydratase-producer, Rhodococcus sp. N-771. The enzyme showed 96.3%, 77.6%, and 30.4% identities with the Oxds of Rhodococcus globerulus A-4, Pseudomonas chlororaphis B23, and Bacillus sp. OxB-1, respectively. The enzyme was expressed in Escherichia coli under the control of the lac- or T7 promoters in its intact and His6-tagged forms, purified, and characterized. The enzyme had heme b as a prosthetic group, catalyzed a stoichiometric dehydration of aldoxime into nitrile, and exhibited the highest activity at neutral pH and at around 30 degrees C similar to the known Oxd from Bacillus sp. OxB-1. The activity was enhanced by reducing agents, such as Na2S, Na2S2(O4), 2-mercaptoethanol, and L-cysteine and supplementary additions of electron acceptors such as flavins, sulfite ion, and vitamin K3. The effect of various chemicals on the enzyme activity was different in the presence and absence of the reducing reagent, Na2S. The enzyme preferentially acts on aliphatic-type substrates and the substrate specificity of the enzyme coincides with that reported for nitrile hydratase produced by the strain. << Less

  J. Biosci. Bioeng. 97:250-259(2004) [PubMed] [EuropePMC]