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The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana

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Abstract

The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943–954, 2013). In the presence of Mg2+ ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. 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. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous dl-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for dl-glycerol-3-phosphate.

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Abbreviations

HAD:

Haloacid dehalogenase-like hydrolase proteins

MBP:

Maltose-binding protein

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Acknowledgments

We acknowledge Professors Montserrat Pagès (CSIC Barcelona, Spain), Thomas Kupke (University of Heidelberg, Germany) and Manuel Hernández (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 Ramón Nogales-Rangel and Alexis González-Policarpo; the Eugenio Grau-Ferrando kind advice and help for sequencing; the Dr Angela Batcheller support in the English edition of the manuscript. 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.

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Authors and Affiliations

  1. 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, 46022, Valencia, Spain

    José A. Caparrós-Martín, Iva McCarthy-Suárez & Francisco A. Culiáñez-Macià

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  1. José A. Caparrós-Martín
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  2. Iva McCarthy-Suárez
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  3. Francisco A. Culiáñez-Macià
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Correspondence to Francisco A. Culiáñez-Macià.

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Caparrós-Martín, J.A., McCarthy-Suárez, I. & Culiáñez-Macià, F.A. The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana . Planta 240, 479–487 (2014). https://doi.org/10.1007/s00425-014-2102-6

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