Purine nucleoside phosphorylase from Pseudoalteromonas sp. Bsi590: molecular cloning, gene expression and characterization of the recombinant protein.
The gene encoding purine nucleoside phosphorylase (PNP) from the cold-adapted marine bacterium Pseudoalteromonas sp. Bsi590 was identified, cloned and expressed in Escherichia coli. The gene encodes a polypeptide of 233 amino acids with a calculated molecular weight of 25,018 Da. Pseudoalteromonas sp. Bsi590 PNP (PiPNP) shares 60% amino sequence identity and conservation of amino acid residues involved in catalysis with mesophilic Escherichia coli deoD-encoded purine nucleoside phosphorylase (EcPNP). N-terminal his-tagged PiPNP and EcPNP were purified to apparent homogeneity using Ni2+-chelating column. Compared with EcPNP, PiPNP possessed a lower temperature optimum and thermal stability. As for PNP enzymes in general, PiPNP and EcPNP displayed complicated kinetic properties; PiPNP possessed higher Km and catalytic efficiency (kcat/Km) compared to EcPNP at 37 degrees C. Substrate specificity results showed PiPNP catalyzed the phosphorolytic cleavage of 6-oxopurine and 6-aminopurine nucleosides (or 2-deoxynucleosides), and to a lesser extent purine arabinosides. PiPNP showed a better activity with inosine while no activity toward pyrimidine nucleosides. The protein conformation was analyzed by temperature perturbation difference spectrum. Results showed that PiPNP had lower conformation transition point temperature than EcPNP; phosphate buffer and KCl had significant influence on PiPNP protein conformation stability and thermostability.
Li
Xiaohui
X
The State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, 200433 Shanghai, China.
Jiang
Xinyin
X
Li
Huirong
H
Ren
Daming
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase