Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica        

Abstract

We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.

Mutagenesis of apyrase conserved region 1 alters the nucleotide substrate specificity

“…10 The specific activity of MP67 for the hydrolysis of ADP is extremely higher than for the hydrolysis of ATP. 7 The biochemical uniqueness of MP67 can be further demonstrated by differences in sensitivity to inhibitors. Apyrases are generally known to be insensitive to inhibitors of P-type, F-type and V-type ATPases; however, MP67 is sensitive to inhibitors of P-type ATPases.…”

“…Apyrases are generally known to be insensitive to inhibitors of P-type, F-type and V-type ATPases; however, MP67 is sensitive to inhibitors of P-type ATPases. 7,10,11 Moreover, although the MP67 enzyme contains the typical apyrase conserved region ACR1-5, comparison of the primary sequences showed that MP67 and MpAPY2 share only 45% identity. Phylogenetic analyses showed that MP67 is a noncanonical apyrases.…”

“…1 All apyrases contain 5 highly conserved apyrase conserved regions (ACR1-5), and exhibit low substrate specificity in the presence of divalent metal ion, i.e., they hydrolyze most nucleoside triphosphates (NTPs) and diphosphates (NDPs). [2][3][4][5][6][7][8][9] We recently reported the presence of two apyrases, MP67 and MpAPY2, in Mimosa pudica. The MP67 is biochemically distinct from other apyrases, and demonstrates substantially higher substrate specificity for ADP than for ATP.…”

“…Phylogenetic analyses showed that MP67 is a noncanonical apyrases. 7 Among the conserved regions, ACR1 and ACR4 are the most important with respect to nucleotide binding. Both ACR1 and ACR4 contain prominent "DXG" two apyrases having different substrate specificity, MP67 and MpaPY2, are present in Mimosa pudica.…”

“…(GenBank ID: AB600997) were ligated into the pCold I expression vector (TaKaRa) as described previously, 7 and were used as templates for site-directed mutagenesis. Serine-63 of MP67 and Ala-75 of MpAPY2 were substituted to Ala and Ser (S63A-MP67 and A75S-MpAPY2), respectively, using the primer pairs (mismatched nucleotides are underlined): 5'-GAT ATT GGA CGC GGG GAG CAC AGG-3' and 5'-CCT GTG CTC CCC GCG TCC AAT ATC-3' for S63A-MP67, and 5'-GTT ATC TTT GAT AGC GGT AGT TCT GG-3' and 5'-CCA GAA CTA CCG CTA TCA AAG ATA AC-3' for A75S-MpAPY2.…”