摘要
Analysis of the amino acid sequence of core protein μuA of avian reovirus has indicated that it may share similar functions to protein μu2 of mammalian reovirus. Since μu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant pA (rμuA) was designed and used to test these activities. μuA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that rμuA possessed NTPase activity that enabled the protein to hydrolyse the β-χ/phosphoanhydride bond of all four NTPs, since NDPs were the only radio labelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k <sub>cat</sub> values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of rμuA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein rμuA The activity of rμuA is dependent on the divalent cations Mg <sup>2+</sup> or Mn <sup>2+</sup> , but not Ca <sup>2+</sup> or Zn <sup>2+</sup> . Optimal NTPase activity of rμuA was achieved between pH 5.5 and 6.0. In addition, rμuA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with rμuA or K408A/K412A rμuA indicated that rμuA, but not K408A/K412A rμuA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of rμuA might be carried out at the same active site, and that protein μuA could play important roles during viral RNA synthesis. © 2007 SGM.