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Applied and Environmental Microbiology, August 2003, p. 4794-4805, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4794-4805.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CynD, the Cyanide Dihydratase from Bacillus pumilus: Gene Cloning and Structural Studies

Dakshina Jandhyala,1 Mark Berman,2,3 Paul R. Meyers,3 B. Trevor Sewell,2 Richard C. Willson,1,4 and Michael J. Benedik1*

Department of Biology and Biochemistry,1 Department of Chemical Engineering, University of Houston, Houston, Texas 77204,4 Electron Microscopy Unit,2 Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa3

Received 2 December 2002/ Accepted 2 May 2003

The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has Km and Vmax values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B. pumilus (strain 8A3) was cloned and expressed. This suggests that cynD, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B. pumilus and is not a reflection of its origin at a mining waste site.

* Corresponding author. Mailing address: Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001. Phone: (713) 743-8377. Fax: (713) 743-8351. E-mail: benedik{at}uh.edu.

Applied and Environmental Microbiology, August 2003, p. 4794-4805, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4794-4805.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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