Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7. 1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD, A new purification procedure for NMN adenylyltransferase from Saccharomyce s cerevisiae provided sufficient amounts of enzyme for tryptic fragmentatio n. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U206 18). The YLR328W gene was isolated, cloned into a T7-based vector and succe ssfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide o f 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypoth etical protein encoded by YLR328W ORF, The N-terminal sequence of the purif ied recombinant NMN adenylyltransferase exactly corresponds to the predicte d sequence. Molecular and kinetic properties of recombinant NMN adenylyltra nsferase are reported and compared with those already known for the enzyme obtained from different sources. (C) 1999 Federation of European Biochemica l Societies.