## 4 Discussion  
The infidelities of molecular recognition of enzymes, which generally exert exquisite specificity, serve as evolutionary starting points of evolvability [38]. Enzyme promiscuity is of great importance in evolutionary biochemistry because it provides opportunities for evolution of new functions in nature [39] and conformational variability is one of the structural principles underlying the evolution of new specificities [14]. Following an earlier work [15], in this study we further investigate sequence determinants underpinning substrate ambiguity. For this purpose, we focused on the evolutionary substrate divergence observed in two members of the HAD wide family of magnesium-dependent acid phosphatases subfamily I with the C1-type cap domain in _Arabidopsis_, the broad-range sugar phosphate phosphatase AtSgpp [19] and the specific DL-glycerol-3-phosphatase AtGpp [18]. Both enzymes exhibit the greatest similarity at the four loops forming the catalytic module of the core domain. Sequence alignment shows the residues forming the catalytic domain to be identical, but not the one of cap domain loop-5. The activity of the core chemistry is common to all subfamilies while loop-5 is tailored toward catalysis of the specialized chemistry unique to a subfamily [20]. What is more, the enzymatic activity within the core domain is dependent on the participation of the residues forming the loop-5 of the corresponding cap domain. We showed that substitution of the putative motif-5 (IAGKH) with that of the DL-glycerol-3-phosphatase AtGpp (MMGRK), does not compromise AtSgpp promiscuousness to the specific targeting of DL-glycerol-3-phosphate [15]. This result suggests that substrate discrimination is not uniquely dependent on the sequence of loop-5 [15]. Intrigued by the former result and because the enhanced substrate promiscuity is often not accompanied by significant changes in the active-site region [40], further structural differences were sought in the cap domain that could determine the dissimilar substrate specificities between both phosphatases. The more evident divergence was observed in the loop motif preceding loop-5, which is extended by three additional residues in AtSgpp. As in previous strategies to investigate the structural basis for the differing substrate specificities in phosphate binding motifs [41], a superimposable mutant was constructed to determine whether the shortening of this loop would constrain the folding plasticity and substrate promiscuity of AtSgpp. As we previously demonstrated, AtSgpp hydrolyzes a wide range of cyclic sugars with noticeable specificity and efficiency (\(k_{\text{cat}}/K_{\text{m}}\) in the range of 2.5-10.7 \(\times\) 10\({}^{3}\) M\({}^{-1}\)s\({}^{-1}\)) [19]. Despite \begin{table}
\begin{tabular}{c c c} \hline \hline
**Substrate** & **AtSgpp** & **AtSgpp3A** \\ \hline  & \(k_{\text{cat}}/K_{\text{m}}\) (M\({}^{-1}\)s\({}^{-1}\)) & \\ D-ribose-5-phosphate & \(1.07\times 10^{4}\) & \(8.3\times 10^{3}\) \\
2-deoxy-D-glucose-6-phosphate & \(7.2\times 10^{3}\) & \(4.9\times 10^{3}\) \\ D-mannose-6-phosphate & \(6.7\times 10^{3}\) & \(2.8\times 10^{3}\) \\ D-glucose-6-phosphate & \(3.4\times 10^{3}\) & \(1.5\times 10^{3}\) \\ D-fructose-6-phosphate & \(2.5\times 10^{3}\) & \(1.2\times 10^{3}\) \\ DL-glycerol-3-phosphate & \(3.1\times 10^{3}\) & \(0.5\times 10^{3}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._this substrate lax specificity, we also observed that AtSgpp could discriminate between isomers that presumably do not accommodate into the active site domain, such as D-glucose-1-phosphate or \(\alpha\)-D-mannose-1-phosphate [19]. Deletion of AtSgpp residues Phe-53, Asn-54, and Asn-55 does not deprive the phosphatase activity of mutant protein AtSgpp3\(\Delta\) but alters the kinetics of the mutated enzyme.