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J. Biol. Chem., Vol. 278, Issue 32, 29600-29608, August 8, 2003
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From the
Institute of Applied Biochemistry and
¶Institute of Materials Science, The University
of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
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.
Received for publication, November 20, 2002 , and in revised form, April 30, 2003.
The nucleotide sequence(s) reported in this paper has been submitted to
the GenBankTM/EBI Data Bank with accession number(s) AB093544
* This work was supported in part by the 21st Century COE (tara.
tsukuba.ac.jp/
coe21/) Program of the Ministry of Education, Culture,
Sports, Science, and Technology; by a grant-in-aid for Scientific Research
from the Ministry of Education, Science, and Culture of Japan; by the
Industrial Technology Research Grant Program in'02 of the New Energy and
Industrial Technology Development Organization of Japan; by the National
Project on Protein Structural and Functional Analyses; and by a Research Grant
(A) of the University Research Projects. The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to the results of this work.
|| To whom correspondence should be addressed. Fax: 81-29-853-4605 (Institute).
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