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Journal of Bacteriology, November 2002, p. 6115-6122, Vol. 184, No. 22
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.22.6115-6122.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Expression, Purification, and Characterization of AknX Anthrone Oxygenase, Which Is Involved in Aklavinone Biosynthesis in Streptomyces galilaeus

Jin-young Chung, Isao Fujii,^* Shigeharu Harada, Ushio Sankawa, and Yutaka Ebizuka

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 28 May 2002/ Accepted 16 August 2002

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His₆ tag was efficiently purified to homogeneity by Ni²⁺ affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.

Present address: Perinatal Research Laboratories, Department of Obstetrics and Gynecology, University of Wisconsin—Madison, Madison, WI 53715.

Present address: International Traditional Medicine Research Center, Toyama International Health Complex, Toyama 939-8224, Japan.

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