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Papers In Press, published online ahead of print May 28, 2003
Institute of Applied Biochemistry, The University of Tsukuba, Tsukuba, Ibaraki 305-8572
Corresponding Author: kobay{at}agbi.tsukuba.ac.jp
Analysis of the nitrile hydratase gene cluster involved in nitrile metabolism of Pseudomonas chlororaphis B23 revealed that it contains one open reading frame (ORF) encoding aldoxime dehydratase upstream of the amidase gene. The amino acid sequence deduced from this ORF shows similarity (32% identity) with that of Bacillus phenylacetaldoxime dehydratase [Biochemistry, 39, 800 (2000)]. 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 on 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.
J. Biol. Chem, 10.1074/jbc.M211832200
Submitted on November 20, 2002
Revised on April 30, 2003
Accepted on May 28, 2003
Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23: Sequencing, gene expression, purification and characterization
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