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Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme
Author(s) -
Emanuelli Monica,
Carnevali Francesco,
Lorenzi Maria,
Raffaelli Nadia,
Amici Adolfo,
Ruggieri Silverio,
Magni Giulio
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00852-2
Subject(s) - nicotinamide mononucleotide , recombinant dna , biochemistry , microbiology and biotechnology , saccharomyces cerevisiae , open reading frame , escherichia coli , biology , enzyme , chemistry , peptide sequence , gene , nad+ kinase , nicotinamide adenine dinucleotide
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 Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. 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 U20618). The YLR328W gene was isolated, cloned into a T7‐based vector and successfully 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 of 48 kDa under SDS‐PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N‐terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.