HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp

[EN] This work presents the isolation and the biochemical characterization of the Arabidopsis thaliana gene AtSgpp. This gene shows homology with the Arabidopsis low molecular weight phosphatases AtGpp1 and AtGpp2 and the yeast counterpart GPP1 and GPP2, which have a high specificity for dl-glycerol-3-phosphate. In addition, it exhibits homology with DOG1 and DOG2 that dephosphorylate 2-deoxy-d-glucose-6-phosphate. Using a comparative genomic approach, we identified the AtSgpp gene as a conceptual translated haloacid dehalogenase-like hydrolase HAD protein. AtSgpp (locus tag At2g38740), encodes a protein with a predicted Mw of 26.7 kDa and a pI of 4.6. Its sequence motifs and expected structure revealed that AtSgpp belongs to the HAD hydrolases subfamily I, with the C1-type cap domain. In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugars substrates (pH optima at 7.0 and K (m) in the range of 3.6-7.7 mM). AtSgpp promiscuity is preferentially detectable on d-ribose-5-phosphate, 2-deoxy-d-ribose-5-phosphate, 2-deoxy-d-glucose-6-phosphate, d-mannose-6-phosphate, d-fructose-1-phosphate, d-glucose-6-phosphate, dl-glycerol-3-phosphate, and d-fructose-6-phosphate, as substrates. AtSgpp is ubiquitously expressed throughout development in most plant organs, mainly in sepal and guard cell. Interestingly, expression is affected by abiotic and biotic stresses, being the greatest under Pi starvation and cyclopentenone oxylipins induction. Based on both, substrate lax specificity and gene expression, the physiological function of AtSgpp in housekeeping detoxification, modulation of sugar-phosphate balance and Pi homeostasis, is provisionally assigned.We acknowledge Professors Montserrat Pages (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernandez (University Polytechnic of Valencia, Spain) for their warm support. We also thank the advice and provision of plasmid pSBETa by Dr. Florence Vignols and Yves Meyer (University of Perpignan, France); the computer software helps by Ramon Nogales-Rangel and Alexis Gonzalez-Policarpo; Eugenio Grau-Ferrando for kind advice and help for sequencing. This work was funded by the 10 month research contract MEC-FEDER to J.A.C.-M., 10 month research contract JAE-DOC to I.M.-S. and by the research project BIO2006-10138 from the MEC-FEDER of Spain to F.A.C.-M. In memoriam of Dr. Mari Cruz Cutanda-Perez.Caparrós Martín, JA.; Mccarthy Suarez, I.; Culiañez Macia, FA. (2013). HAD hydrolase function unveiled by substrate screening: enzymatic characterization of Arabidopsis thaliana subclass I phosphosugar phosphatise AtSgpp. Planta. 237(4):943-954. https://doi.org/10.1007/s00425-012-1809-59439542374Allen KN, Dunaway-Mariano D (2004) Phosphoryl group transfer: evolution of a catalytic scaffold. 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