The similarities in sequence amongst eukaryotic and bacteriophage phosphatases in Figure 2 suggests that they share a common catalytic mechanism. Several PP1, PP2A and PP2B homologues in different species [2] and the novel phosphatases, PP4 (formerly PPX) [41] and PP5 (M. X. Chen, Y.H. Chen and P.T.W. Cohen, in preparation), have been shown to dephosphorylate serine and threonine residues in proteins. The lambda bacteriophage phosphatase ORF221 has been demonstrated to possess protein phosphatase activity against glycogen phosphorylase a and PKA phosphorylated casein, [42]. When expressed from a recombinant vector, ORF221 displays phosphatase activity against casein phosphorylated on serine and tyrosine residues [43]. Consequently the 15 residues (Table 1) invariant throughout the eukaryotic and bacteriophage phosphatases are likely to play crucial roles in protein folding and enzyme catalysis.
The novel phosphatases PPQ, PPT, PPV, PPY, PPZ, PPH3, SIT4 have not yet been demonstrated to be active phosphatases. However, virtually all residues that are invariant between PP1, PP2A , PP2B, PP4 and PP5 are also invariant between PPQ, PPT, PPV, PPY, PPZ, PPH3 and SIT4. The exception, serine, is invariant at position 192 for all sequences except PPT where it is a proline, but since serine is not conserved in the bacteriophage sequences it is unlikely to be an essential residue. The E. coli tetra-phosphatase removes pyrophosphate from diadenosine tetra-phosphate. Since 14 of the 15 residues, that are invariant throughout eukaryote and bacteriophage phosphatases, are also invariant in E. coli tetra-phosphatase (Table 2), it is likely that the E. coli phosphatase shares a common catalytic mechanism with the eukaryotic and bacteriophage enzymes. It is not known whether E. coli diadenosine tetraphosphatase possesses protein phosphatase activity.
The importance of the invariant residues, Asp 59 and 88 and His 61 and 139 in catalysis is supported recent site directed mutagenesis study of lambda bacteriophage phosphatase ORF221, (J. E. Dixon and S. Zhou, unpublished results). Substitution of Asp 59 for Asn, His 61 for Asp, Asp 88 for Asn and His 139 for Asn abolishes phosphatase activity. In contrast substitution of either Asp 91 for Asn and Glu 140 for Gln reduces activity by 29%and 82%, respectively. Although Asp 91 and Glu 140 are invariant throughout eukaryotic and bacteriophage sequences, Asp 91 is an Ala and Glu 140 is an Asp in the E. coli diadenosine tetra-phosphatase sequence, consistent with the notion that these residues are not essential for phosphatase activity.
There are no invariant Arg, Lys, His, Asp or Glu residues after position 296 or before position 58 of the eukaryotic phosphatases (Figure 1). Moreover, before position 58, the sequences of the phage and E. coli phosphatases show little similarity to the eukaryotic proteins (Figure 2). Together, this suggests that neither of these regions are likely to be involved directly in either phosphate binding or metal ion binding.