Extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Gene cloning and amino acid sequence determination.
A gene encoding an extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase was cloned from the archaeon Sulfolobus solfataricus. Two degenerate oligodeoxyribonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the protein were used to screen a genomic library of S. solfataricus cloned into the pGEM7Zf(+) vector. The DNA fragment of 2118 bp containing the 5'-methylthioadenosine phosphorylase gene was sequenced. The open reading frame comprises 711 nucleotides, which includes the stop codon, and encodes a protein of 236 residues whose molecular mass is in good agreement with the value determined by gel filtration for the purified enzyme. The N- and C-terminal sequences of the protein and the sequences of the peptides prepared by cyanogen bromide cleavage exactly match with the corresponding sequences deduced from the gene, thus confirming the identity of the 5'-methylthioadenosine phosphorylase gene. Typical archaebacterial regulatory sites were identified in the flanking regions and a potential Shine-Dalgarno-like sequence was recognized around the ATG initiation codon. The deduced amino acid sequence showed 32% identity and 30% identity with Escherichia coli purine-nucleoside phosphorylase and with E, coli uridine phosphorylase, respectively. Evolutionary and structural implications of this similarity are discussed.
Cacciapuoti
G
G
Istituto di Biochimica delle Macromolecole, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Italy.
Porcelli
M
M
Bertoldo
C
C
Fusco
S
S
De Rosa
M
M
Zappia
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase