To test the effect of amino acid substitutions on tPphA activity, the various phosphatase variants were assayed with the following substrates: the artificial substrate p-nitrophenyl phosphate (pNPP) (Table 1), three different phosphopeptides, of which one corresponds to the phosphorylated T-loop of PII (Table 2), and the physiological substrate PII-P (Fig. 3 and Fig. S1A–C). As shown previously, the alanine variants of the aspartate residues D18, D34, D119, D193 and D231, which coordinate the metals in the catalytic center, are catalytically inactive . The newly generated variants displayed a large heterogeneity with respect to their catalytic properties, as described in more detail in the following. Figure 4A shows a comprehensive comparison of the variants, which were differentially affected in substrate specificity. To facilitate the comparison, the enzyme activities towards the five different substrates were normalized to the respective activity of wild-type tPphA, which was set to 100%. Among all newly created variants, variant Q17K was most strongly affected in catalytic activity, with only about 10% residual activity towards pNPP and the threonyl peptide and no detectable activity towards seryl peptides and PII-P. The tPphA crystal structure (PDB file 2J82 ) reveals that Gln17 indirectly coordinates M2 by a bridging water molecule. In many other PP2C homologs, the corresponding position is occupied by a glutamyl residue (see Supplementary file S1 in ). Interestingly, the Gln17 to Glu mutation (Q17E) resulted in increased catalytic efficiency towards an artificial phosphoseryl peptide (pS-peptide), whereas the activity towards the other substrates was very similar to wild-type tPphA. Of the other novel variants, variant S15A was not significantly affected in catalytic properties. Several variants (R13K, M36A, T138A, R160A, H161A and R169A) showed a general reduction in catalytic activity, dephosphorylating all tested substrates more slowly than wild-type tPphA. Apparently, these point mutations affected the catalytic activity of the corresponding tPphA variants to different degrees, showing effects on Kcat and Km as presented in Table 1. Residues Arg13 and Met36 are close to the catalytic center of tPphA. The conservative replacement of Arg13 by Lys resulted in a moderate reduction of activity. By contrast, the M36A variant showed strongly reduced catalytic activity towards all substrates, suggesting that the bulky methionyl side chain could be required to stabilize the structure of the catalytic center or to exclude water from the catalytic center. Alanine replacement of residues Thr138, Arg160, His161 and Arg169, all belonging to the flap subdomain (Figs 1A and 2A), moderately reduced enzyme activity towards all tested substrates. This effect on catalysis could be due to subtle flap subdomain interactions with the catalytically active third metal (M3) as shown in previous structures [20,21]. Finally, two variants, H39A and T138E, were differentially affected in substrate reactivity (Fig. 4A). These two variants could dephosphorylate artificial substrates but showed almost no activity towards PII-P. Alanine replacement of residue His39, which is highly conserved in bacterial PP2C family members (H39A), did not at all impair the reactivity towards pNPP, but, intriguingly, this variant displayed reduced activity towards artificial phosphopeptides and was almost completely unable to dephosphorylate PII-P. The undisturbed activity towards pNPP clearly indicates that this peripheral part of the enzyme does not directly take part in catalysis. The impaired reactivity towards phosphopeptides and phosphoprotein implies a role of His39 in substrate specificity. Mutation of Thr138 to Glu (T138E) resulted in a surprisingly high disturbance of activity. This was unexpected, since in numerous bacterial PP2C members the corresponding position is in fact occupied by Asp or Glu residues (see Supplementary file S1 in ). The methyl group of Thr138 projects into the basis of the flap subdomain and introduction of a negative charge at this place may distort the conformation of the entire flap subdomain region. A closer look at the data shows that the enzyme lost about 80% of activity when assayed with pNPP; it was even less active towards the artificial pS- and pT-phosphopeptides and was almost completely inactive towards phospho-PII. Strikingly, approximately 50% activity was regained with the T-loop peptide as substrate. Therefore, in addition to general damage of the catalytic activity, the T138E mutation seems to affect substrate specificity.
Figure 3. (A) Graphical representation of PII-P dephosphorylation assays over a period of 45 min analyzed by non-denaturing gels, shown in Fig. S1A, for tPphA wild-type (WT) and variants S15A, Q17E, T138A, R13K, R169A and H161A. (B) Graphical representation of PII-P dephosphorylation assays over a period of 90 min analyzed by non-denaturing gels, shown in Fig. S1B, for tPphA variants H161A, R160A, H39A, M36A, T138E, Q17K and D193A.
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The activities of human PP2Cα and chimera A (tPphA catalytic core + human PP2Cα flap) were also assayed towards the above mentioned five different substrates. Human PP2Cα dephosphorylated artificial substrates faster than wild-type tPphA but was unable to dephosphorylate PII-P (Tables 1 and 2 and Fig. S1C). The inability to dephosphorylate PII-P is not due to an unfavorable amino acid context around phosphoseryl residue 49, since the phosphopeptide corresponding to the phosphorylated T-loop of PII is readily dephosphorylated. To prove that the flap subdomain of human PP2Cα is responsible for the specific exclusion of PII-P as substrate, the flap was grafted on the catalytic core of tPphA (chimera A). The resulting hybrid protein retained indeed some residual activity; however, it could only dephosphorylate pNPP to some extent (17% residual activity) but could not dephosphorylate phosphopeptides and PII-P (Tables 1 and 2 and Fig. 4B), indicative of an injured enzyme. The catalytic properties in the pNPP dephosphorylation assay showed that the hybrid enzyme was strongly affected for Kcat, whereas the Km values for pNPP and Mn2+ were very similar to the respective Km values of wild-type tPphA (Table 1). In agreement with these properties, isothermal titration calorimetry analysis of metal binding by chimera A revealed that chimera A bound Mn2+ with a similar affinity to wild-type tPphA (Fig. S2 and Table S1). These results imply that chimera A is able to form a metal cluster which is partly functional, but access of the substrates to the catalytic site is severely hampered in the hybrid protein. Only the smallest substrate, pNPP, can be processed in the catalytic site, albeit with reduced kinetics.