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PMID:12902273
| Citation |
Jandhyala, D, Berman, M, Meyers, PR, Sewell, BT, Willson, RC and Benedik, MJ (2003) CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies. Appl. Environ. Microbiol. 69:4794-805 |
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| Abstract |
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 K(m) and V(max) 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. |
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| Keywords |
Amino Acid Sequence; Bacillus/classification; Bacillus/enzymology; Bacterial Proteins/chemistry; Base Sequence; Cloning, Molecular; Cyanides/metabolism; Escherichia coli/genetics; Hydrogen-Ion Concentration; Hydrolases/chemistry; Hydrolases/genetics; Hydrolases/physiology; Image Processing, Computer-Assisted; Microscopy, Electron; Molecular Sequence Data; Phylogeny; Protein Structure, Quaternary |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0000257: nitrilase activity |
ECO:0000314: |
F |
In text on page 4802 "This gene was subcloned into pET26b and transformed into E. coli BL-21(DE3) pLysS for expression. Following induction, cell lysates from this strain (MB3033) had cyanide-degrading activity comparable to lysates of MB2890 and MB2899, sug- gesting that although the cyanide dihydratase gene is func- tional in B. pumilus 8A3, it most likely is not expressed; how- ever, formally, it could merely be regulated differently than cynD of B. pumilus C1 or could be coexpressed with an inhib- itor." |
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