Abstract
The Arabidopsis thaliana broad-range sugar phosphate phosphatase AtSgpp (NP_565895.1, locus AT2G38740) and the specific DL-glycerol-3-phosphatase AtGpp (NP_568858.1, locus AT5G57440) are members of the wide family of magnesium-dependent acid phosphatases subfamily I with the C1-type cap domain haloacid dehalogenase-like hydrolase proteins (HAD). Although both AtSgpp and AtGpp have a superimporsable α/β Rossmann core active site, they differ with respect to the loop-5 of the cap domain, accounting for the differences in substrate specificity. Recent functional studies have demonstrated the essential but not sufficient role of the signature sequence within the motif-5 in substrate discrimination. To better understand the mechanism underlying the control of specificity, we explored additional sequence determinants underpinning the divergent evolutionary selection exerted on the substrate affinity of both enzymes. The most evident difference was found in the loop preceding the loop-5 of the cap domain, which is extended in three additional residues in AtSgpp. To determine if the shortening of this loop would constrain the substrate ambiguity of AtSgpp, we deleted these three aminoacids. The kinetic analyses of the resulting mutant protein AtSgpp3Δ (ΔF53, ΔN54, ΔN55) indicate that promiscuity is compromised. AtSgpp3Δ displays highest level of discrimination for D-ribose-5-phosphate compared to the rest of phosphate ester metabolites tested, which may suggest a proper orientation of D-ribose-5-phosphate in the AtSgpp3Δ active site.
Highlights
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]
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 greatest similarity occurs at the four motifs forming the catalytic scaffold of the active site platform framed by the core domain [36], which contains the highly conserved sequence motifs by which family members are recognized (Figure 1, motifs 1, 2, 3, and 4)
Summary
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]. 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]. 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 α/β core domain catalytic scaffold, in Biology 2019, 8, 77; doi:10.3390/biology8040077 www.mdpi.com/journal/biology
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