Characterization of nicotinamide mononucleotide adenylyltransferase from thermophilic archaea.
The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of NAD+ and nicotinic acid adenine dinucleotide. It has been purified to homogeneity from cellular extracts of the thermophilic archaeon Sulfolobus solfataricus. Through a database search, a highly significant match was found between its N-terminal sequence and a hypothetical protein coded by the thermophilic archaeon Methanococcus jannaschii MJ0541 open reading frame (GenBank accession no. U67503). The MJ0541 gene was isolated, cloned into a T7-based vector, and expressed in Escherichia coli cells, yielding a high level of thermophilic NMN adenylyltransferase activity. The expressed protein was purified to homogeneity by a single-step chromatographic procedure. Both the subunit molecular mass and the N-terminal sequence of the pure recombinant protein were as expected from the deduced amino acid sequence of the MJ0541 open reading frame-encoded protein. Molecular and kinetic properties of the enzymes from both archaea are reported and compared with those already known for the mesophilic eukaryotic NMN adenylyltransferase.
Raffaelli
N
N
Istituto di Biochimica, Facoltà di Medicina e Chirurgia, Università di Ancona, Italy.
Pisani
F M
FM
Lorenzi
T
T
Emanuelli
M
M
Amici
A
A
Ruggieri
S
S
Magni
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology*
D000596:Amino Acids; D019843:Archaeal Proteins; D011994:Recombinant Proteins; D009612:Nicotinamide-Nucleotide Adenylyltransferase