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Sequence Determinants of Substrate Ambiguity in a HAD Phosphosugar Phosphatase of Arabidopsis Thaliana

Instituto de Biología Molecular y Celular de Plantas ‘‘Eduardo Primo Yúfera’’ (UPV-CSIC), Universidad Politécnica de Valencia, Ciudad Politécnica de la Innovación (CPI), C/ Ingeniero Fausto Elio s/n, ES-46022 Valencia, Spain
CHIRI Research Centre, Faculty of Health Sciences, School of Pharmacy and Biomedical Sciences, Curtin University, Perth 6102, WA, Australia
Author to whom correspondence should be addressed.
Biology 2019, 8(4), 77;
Received: 22 July 2019 / Revised: 17 September 2019 / Accepted: 6 October 2019 / Published: 9 October 2019
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. View Full-Text
Keywords: Arabidopsis; HAD superfamily; hydrolases; sugar phosphatases; promiscuity Arabidopsis; HAD superfamily; hydrolases; sugar phosphatases; promiscuity
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Caparrós-Martín, J.A.; McCarthy-Suárez, I.; Culiáñez-Macià, F.A. Sequence Determinants of Substrate Ambiguity in a HAD Phosphosugar Phosphatase of Arabidopsis Thaliana. Biology 2019, 8, 77.

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