Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae.
The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD(+). On the basis of a remarkable structural similarity with previously described Saccharomyces cerevisiae NMNAT (yNMNAT-1), the YGR010-encoded protein was identified as a second isoform of yeast NMNAT (yNMNAT-2). The YGR010 gene was isolated, cloned into a T7-based vector, and successfully expressed in Escherichia coli BL21 cells, yielding high level of NMN adenylyltransferase activity. The purification procedure reported in this paper, consisting of two chromatographic steps, allowed the isolation of 3mg of electrophoretically homogeneous yNMNAT-2 from 1 liter of E. coli culture. Under SDS/PAGE, the recombinant protein resulted in a single polypeptide of 46 kDa, in agreement with the molecular mass of the hypothetical protein encoded by YGR010 gene. The N-terminal sequence of the purified recombinant yNMNAT-2 exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant yNMNAT-2 are reported and compared with those already known for yNMNAT-1.
Emanuelli
Monica
M
Facoltá di Medicina, Istituto di Biochimica, Università di Ancona, via Ranieri, 60100 Ancona, Italy.
Amici
Adolfo
A
Carnevali
Francesco
F
Pierella
Francesca
F
Raffaelli
Nadia
N
Magni
Giulio
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009537:Nicotinamide Mononucleotide / Q000378:metabolism; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000235:genetics*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D020033:Protein Isoforms; D011994:Recombinant Proteins; D009243:NAD; D009537:Nicotinamide Mononucleotide; D000255:Adenosine Triphosphate; D009612:Nicotinamide-Nucleotide Adenylyltransferase