## 1 Introduction  
Molecular biology has evolved from the central dogma of gene-protein-specific function [1; 2] and the analogy of specific substrates suiting the binding pocket lock [3]. Thus, substrate specificity is considered the consequence of the divergent evolutionary selection exerted on the substrate promiscuity activity of precursor enzymes [4; 5]. The ability of enzymes to accommodate different substrates is known as substrate ambiguity [6]. Substrate ambiguous enzymes require active site plasticity [7; 8] and the involvement of additional functional residues besides those essential for core catalytic activity [9; 10; 11; 12; 13]. Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions [14]. In an earlier work [15], we investigated the determinants of substrate promiscuity focusing on the evolutionary divergence of two _Arabidopsis_ members of the haloacid dehalogenase-like hydrolase proteins (HAD) superfamily. The members of this family share a number of structural features including the \(\alpha\)/\(\beta\) core domain catalytic scaffold, in which the active site consists of four loop motifs containing highly conserved residues. Motifs one (DxD), two (T/S) and three (K/R) are less flexible than motif 4, which has been variously described (E/DD, GDxxxD\(\times\times\times\)D or GDxxxD). Spatially, these four structural modules are arranged around the substrate-binding groove, which positions in the active site architecture the key residues involved in the core chemistry [16]. Many of the HAD family members also contain a smaller module connected to the core domain by two hinge loops. This module known as cap domain, acts as a lid over the core catalytic domain. The cap domain localizes residues that are important for the enzymatic catalysis such as those contained in loop-5. This loop locates a stringently conserved Gly residue flanked by amino acids whose side chains contribute to the catalytic site formed at the domain-domain interface. While the \(\alpha\)/\(\beta\) Rossmann fold structural domains are superimposable, the configuration of the cap domain is less conserved even considering the closest structural homologs [17]. _Arabidopsis thaliana_ AtGpp (NP_568858.1) [18] and AtSgpp (NP_565895.1) [19] are both HAD-type phosphosugar phosphatases. Structural prediction and chemistry analysis classify them as typical phosphomonoesterases of subclass I (C1-type cap) [19]. Although both enzymes share significant similarity (39%, sequence identity of 23%) and their predicted tertiary structures are superimposable, their substrate specificity is different. AtGpp exhibits a specific preference for the hydrolysis of DL-glycerol-3-phosphate, while AtSgpp is a promiscuous enzyme that shows phosphate phosphatase activity over a broad-range of sugar substrates, including intermediates of the pentose phosphate and glucose metabolism such as D-glucose-6-phosphate, D-ribose-5-phosphate, D-fructose-6-phosphate, or DL-glycerol-3-phosphate, amongst others [18; 19]. The structural motifs of the core catalytic domain of both AtSgpp and AtGpp proteins present the same amino acid composition [16; 20]. Conversely, large differences are observed in the residues forming the substrate specificity region (loop-5) of the cap domain [16]. Residues in the loop-5, are responsible for the substrate diversification within the HAD family and provide substrate specificity [20; 21]. However, the substitution of the entire motif-5 in AtSgpp (IAGKH) with that of AtGpp (MMGRK) does not restrict AtSgpp activity to the DL-glycerol-3-phosphate [15]. The lack of activity of the resultant AtSgpp mutant enzyme (I68M, A69M, K71R, H72K) lead us to hypothesize a more complex chemistry in which, apart from those of the sequence motif-5, the side chains of other residues of the cap domain would be involved [16].