A. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in E. coli. A cAMP-independent variant, in which alanine-144 is replaced by threonine (CAP-91), is activated by adenosine, which does not activate the wild-type CAP. To test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of adenosine and then X-ray diffraction data were accumulated. The data from the difference Fourier map suggested that adenosine becomes bound to one of the two subunits of CAP, while the other remains occupied by cAMP. B. Effect of Site-specific Mutations in HPr on the Gram-positive Phosphoenolpyruvate:sugar Phosphotransferase System (PTS). The PTS is regulated in gram-positive bacteria by a protein kinase mediated phosphorylation of serine-46 of the phosphocarrier protein known as HPr. To define the mechanism of the regulation, site-specific mutations in serine-46 of HPr were constructed and then tested for PTS activity. The results indicate that imposition of a negative charge on serine-46 of HPr markedly inhibits its activity as a phosphocarrier. C. Hyperexpression and Purification of Escherichia coli Adenylate Cyclase Using a Vector Designed for Expression of Lethal Gene Products. Since high levels of cAMP are toxic to E. coli, difficulties have been experienced in constructing strains that overproduce the enzyme adenylate cyclase, responsible for the synthesis of cAMP. A plasmid vector suitable for the expression of lethal genes was therefore constructed. Using a derivative of this vector, containing the gene for adenylate cyclase, it was possible to increase the cellular level of the enzyme about 7000 fold, corresponding to 30% of the total protein. A relatively simple procedure was devised to purify adenylate cyclase from extracts after hyperexpression to yield a nearly homogenous protein.
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