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J. Biol. Chem., Vol. 279, Issue 30, 31171-31176, July 23, 2004

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Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme^*

Dan Gu, Yuzhen Zhou¶, Verena Kallhoff||, Berevan Baban**, John J. Tanner, and Donald F. Becker¶

From the Department of Chemistry and Biochemistry, University of Missouri, St. Louis, Missouri 63121, the ^¶Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, and the Departments of Chemistry and Biochemistry, University of Missouri, Columbia, Missouri 65211

The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1–261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1–90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nM, which is slightly higher than that of the PutA-DNA complex (K_d 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-helix DNA binding motif in residues 1–47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nM for the PutA47-DNA complex. Thus, N-terminal residues 1–47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.

Received for publication, April 2, 2004 , and in revised form, May 17, 2004.

^* This work was supported in part by National Science Foundation Grant MCB0091664 and by National Institutes of Health grants GM61068 (to D. F. B.) and GM65546 (to J. J. T.), the University of Nebraska Biochemistry Department and Redox Biology Center, and the Nebraska Agricultural Research Division (Journal Series no. 14606). This publication was also made possible by National Institutes of Health Grant P20-RR-017675-02 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. 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.

Current address: Dept. of Chemistry, Washington University, Campus Box 1134, One Brookings Dr., St. Louis, MO 63130.

^|| Current address: Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

^** Current address: Dept. of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8108, St. Louis, MO 63110.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Nebraska, N258 Beadle Center, Lincoln, NE 68588; Tel.: 402-472-9652; Fax: 402-472-7842; E-mail: dbecker3{at}unl.edu.

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