The incorporation and distribution of 14C in 2'-deoxycoformycin, elaborated by Streptomyces antibioticus, were studied with [U-14C]glycine, [U-14C]adenosine and [U-14C]adenine. Similar ratios of 14C in the aglycon and carbohydrate portions of 2'-deoxycoformycin, ara-A, and adenosine isolated from the RNA indicated that [U-14C]adenosine was incorporated into 2'-deoxycoformycin without cleavage of the N-glycosylic bond. Following the addition of [U-14C]adenine, 98% of the 14C isolated from [14C]-2'-deoxycoformycin resided in the aglycon. 2'-Deoxycoformycin biosynthesis may not require the de novo purine biosynthetic pathway as evidenced by the failure to detect incorporation of [U-14C]glycine into 2'-deoxycoformycin. These data suggest that the biosynthesis of 2'-deoxycoformycin involves the incorporation of the carbon-nitrogen skeleton of an intact purine nucleoside or nucleotide, thereby implying that a purine ring is opened enzymatically between C-6 and N-1 and a one-carbon unit is added to form the 1,3-diazepine ring of 2'-deoxycoformycin.
The gene nirM, coding for cytochrome c-551 in Pseudomonas stutzeri substrain ZoBell, was engineered to mutate Met61, the sixth ligand to the heme c, into His61, thereby converting the typical Met-His coordination of a c-type cytochrome into His-His, typical of b-type cytochromes. The mutant protein was expressed heterologously in Escherichia coli at levels 3-fold higher than in Pseudomonas and purified to homogeneity. The mutant retained low-spin visible spectral characteristics, indicating that the strong field ligand His 61 was coordinated to the iron. The physiochemical properties of the mutant were measured and compared to the wild-type properties. These included visible spectra, ligand binding reactions, stability to temperature and chemical denaturant, oxidation-reduction potentials, and electron-transfer kinetics to the physiological nitrite reductase of Pseudomonas. Despite a change in potential from the normal 260 mV to 55 mV, the mutant retained many of the properties of the c-551 family.
The polymorphic ciliated protozoan Tetrahymena vorax can undergo differentiation from the microstomal form, which normally feeds on bacteria and other particulate matter, into the macrostomal cell type, which is capable of ingesting prey ciliates. The process is triggered by exposure of the microstome to an inducer contained in stomatin, an exudate of the prey. To establish the identity of the signal, stomatin was fractionated by combinations of cation exchange, HPLC, and TLC, and the fractions were assayed for biological activity. Although no single active fraction of purified inducer was obtained, all fractions with activity contained ferrous iron and the nucleic acid catabolites hypoxanthine (6-oxypurine) and uracil (2,4-dioxopyrimidine), probably in a chelated form. The activity of synthetic complexes containing these three components is equivalent to stomatin. These results indicate a role for ferrous iron and its potential in chelated form to signal differentiation in certain protozoa and, perhaps, in other organisms as well.
In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase a subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein.
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