The gene encoding streptococcal NAD^+-glycohydrolase : datermination of primary structure and analysis of enzymatic active sites

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1. Cloning of a DNA fragment corresponding to the N-terminus amino acid sequence of streptoccal NAD^+-glycohydrolaseAn N-terminus amino acid sequence of streptococcal NAD^+-glycohydrolase was determined by using the purified enzyme. To amplify a DNA fragment which corresponded to the N-terminus amino acid sequence, PCR-primers were designed and PCR was done. A DNA fragment which had the expected size (120 bp) was amplified. The nucleotide and the deduced amino acid sequences of this fragment revealed that this fragment was a part of the gend encoding NAD^+-glycohydrolase.2. Primary structure of the gene encoding NAD^+-glycohydrolase and construction of its expression systemSouthern blot analysis was done using the 120-bp DNA fragment. It was shown that the gene encoding NAD^+-glycohydrolase was contained as a single copy in chromosome. Plasmid libraries of chromosomal DNA from Streptococcus pyogenes C2556 were constructed. Single specific-PCR was preformed using primers designed from sequences of the plasmid and of the 120-bp DNA fragment. The nucleotide sequence of the amplicon was determined. The open reading frame was ca.1400 bp. Calculated molecular weight and pI were ca.50,000 and 8.5, respectively. These properties were consistent with those of the purified enzyme. Full length of the gene encoding NAD^+-glycohydrolase was cloned and expressed in Escherichia coli. Cell extracts showed not only NAD^+-glycohydrolase activity bu also cyclic ADP-ribose-synthesis and -hydrolysis activities. Enzymatic active portions are currently under investigation.