ABSTRACT
The cDNA for the human trifunctional enzyme of de novo purine biosynthesis, which encodes glycinamide ribonucleotide synthetase, aminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide trans-formylase, has been overexpressed in Escherichia coli and its protein product has been purified to homogeneity. The glycinamide ribonucleotide transformylase activity, which constitutes the C-terminal domain of the trifunctional enzyme, has been characterized with respect to its kinetic constants, Vmax = 3.03 +/- 0.15 micromol/min-mg and Km values for beta-glycinamide ribonucleotide and 10-formyl-5,8-dideazafolate of 0.94 +/- 0.21 and 1.58 +/- 0.25 microM, respectively, and its kinetic mechanism, which is ordered-sequential with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.
Subject(s)
Carbon-Nitrogen Ligases/genetics , Carbon-Nitrogen Ligases/isolation & purification , Hydroxymethyl and Formyl Transferases/genetics , Hydroxymethyl and Formyl Transferases/isolation & purification , Purines/biosynthesis , Carbon-Nitrogen Ligases/metabolism , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Humans , Hydroxymethyl and Formyl Transferases/metabolism , Kinetics , Oligodeoxyribonucleotides/genetics , Phosphoribosylglycinamide Formyltransferase , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
The Sth132I restriction endonuclease (R.Sth132I) was detected in Streptococcus thermophilus ST132 and purified to near homogeneity by heparin Sepharose CL-6B affinity chromatography. Fragments from Sth132I digestion of plasmid DNA were subcloned into pUC19 in Escherichia coli DH5alpha and sequenced. Sequence analysis of inserts and their ligation junction sites revealed that Sth132I is a novel class-IIS restriction endonuclease, which recognizes the non-palindromic sequence 5'-CCCG(N)4-3', 3'-GGGC(N) 8-5'.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/isolation & purification , Deoxyribonucleases, Type II Site-Specific/metabolism , Streptococcus/enzymology , Base Sequence , Chromatography, Affinity , Cloning, Molecular , DNA/genetics , DNA/metabolism , Escherichia coli/genetics , Molecular Sequence Data , Plasmids/genetics , Plasmids/metabolism , Recombination, Genetic , Sequence Analysis, DNAABSTRACT
A new HaeIII isoschizomer from Streptococcus agalactiae was isolated by a single-step purification method. The highly active restriction endonuclease, SagI, was free of nonspecific nuclease activity and was suitable for use in molecular biology procedures. The rapid isolation procedure may be applicable for the recovery of other restriction endonucleases from bacteria.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/isolation & purification , Streptococcus agalactiae/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Substrate SpecificityABSTRACT
Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (Km = 7.0 microM) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (Km = 15.3 microM). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.
