| Autor: |
Nesbitt NM; Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400 United States., Arora DP; Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400 United States., Johnson RA; Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400 United States., Boon EM; Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400 United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Biotechnology reports (Amsterdam, Netherlands) [Biotechnol Rep (Amst)] 2015 May 05; Vol. 7, pp. 30-37. Date of Electronic Publication: 2015 May 05 (Print Publication: 2015). |
| DOI: |
10.1016/j.btre.2015.04.008 |
| Abstrakt: |
Cyclic-diGMP is a bacterial messenger that regulates many physiological processes, including many attributed to pathogenicity. Bacteria synthesize cyclic-diGMP from GTP using diguanylate cyclases; its hydrolysis is catalyzed by phosphodiesterases. Here we report the over-expression and purification of a bi-functional diguanylate cyclase-phosphodiesterase from Agrobacterium vitis S4. Using homology modeling and primary structure alignment, we identify several amino acids predicted to participate in the phosphodiesterase reaction. Upon altering selected residues, we obtain variants of the enzyme that efficiently and quantitatively catalyze the synthesis of cyclic-diGMP from GTP without hydrolysis to pGpG. Additionally, we identify a variant that produces cyclic-diGMP while immobilized to NiNTA beads and can catalyze the conversion of [α- 32 P]-GTP to [ 32 P]-cyclic-diGMP. In short, we characterize a novel cyclic-diGMP processing enzyme and demonstrate its utility for efficient and cost-effective production of cyclic-diGMP, as well as modified cyclic-diGMP molecules, for use as probes in studying the many important biological processes mediated by cyclic-diGMP. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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