Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus.
An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 degrees C revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose.
Sacchetti
Silvana
S
Dipartimento di Chimica Biologica, Università degli Studi di Napoli "Federico II", Naples, Italy.
Bartolucci
Simonetta
S
Rossi
Mosè
M
Cannio
Raffaele
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D019642:RNA, Archaeal / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
D019641:DNA, Archaeal; D008360:Mannosephosphates; D019642:RNA, Archaeal; D011993:Recombinant Fusion Proteins; C047217:mannose 1-phosphate; D006155:Guanosine Diphosphate Mannose; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase