Purification and characterization of GTP cyclohydrolase I from Streptomyces tubercidicus, a producer of tubercidin

GTP cyclohydrolase I catalyzing the first reaction in the biosynthesis of p terin moiety of folic acid in bacteria, was purified from Streptomyces tube rcidicus by at least 203-fold with a yield of 32% to apparent homogeneity, using ammonium sulfate fractionation, DEAE-cellulose, Sepharose CL-GB, and hydroxylapatite column chromatography. The molecular weight of the native e nzyme was estimated to be 230,000 daltons by gel permeation chromatography. The purified enzyme gave a single band on sodium dodesyl sulfate-poIyacryl amide gel electrophoresis and its molecular weight was apparently 58,000 da ltons. These results indicate that the enzyme consists of four subunits wit h the same molecular weight. The K-m and V-max values for CTP of the purifi ed enzyme were determined to be 80 mu M and 90 nmol/min (mg protein), respe ctively. The optimum pH and temperature for the enzyme reaction were pH 7. 5 similar to 8.5 and 40 similar to 42 degrees C, respectively. Coenzyme or metal ion was not required for the enzyme activity. The enzyme activity was inhibited by most divalent cations, while it was slightly activated by pot assium ion. In case of nucleotides, CTP, CMP, GDP, and UTP inhibited enzyme activity, among which GDP exhibited the strongest inhibitory effect.