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Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

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Abstract

Phosphoribosyl pyrophosphate synthetase (PRS—EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

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Abbreviations

PRS:

phosphoribosyl pyrophosphate synthetase

SUCEST:

Sugar Cane EST Genome Project

EST:

expressed sequence tag

PRPP:

5-phosphoribosyl-α-1-pyrophosphate

IPTG:

isopropyl β-d-1-thiogalactopyranoside

AMP:

adenine ribonucleotide monophosphate

ATP:

adenine ribonucleotide triphosphate

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Acknowledgments

This work was supported in part by a research grant 99/02874-9 to O.H. Thiemann (FAPESP). H.B. Napolitano receives scholarship from FAPESP. We would like to thank the members of the Protein Crystallography and Structural Biology and Biophysics Groups (IFSC-USP) for helpful discussions in the course of this work. We are grateful to Professor Lewis Joel Greene and Dr. Vitor Marcel Faça of the Protein Chemistry Centre, Medicine School of Ribeirão Preto, for the amino-terminal sequencing of the PRS fragments.

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Authors and Affiliations

  1. Departmento de Genética e Evolução, Instituto de Física de São Carlos, Universidade de São Paulo, Cx. Postal 369, 13566-590, São Carlos, SP, Brazil

    Susana Andrea Sculaccio, Hamilton Barbosa Napolitano, Leila Maria Beltramini, Glaucius Oliva & Otavio Henrique Thiemann

  2. Instituto de Química de São Carlos Universidade de São Paulo, Cx. Postal 780, 13566-590, São Carlos, SP, Brazil

    Emanuel Carrilho

  3. Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, BR 153 Km 18, 75001-970, Anápolis, GO, Brazil

    Hamilton Barbosa Napolitano

Authors
  1. Susana Andrea Sculaccio
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  2. Hamilton Barbosa Napolitano
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  3. Leila Maria Beltramini
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  4. Glaucius Oliva
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  5. Emanuel Carrilho
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  6. Otavio Henrique Thiemann
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Correspondence to Otavio Henrique Thiemann.

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Sculaccio, S.A., Napolitano, H.B., Beltramini, L.M. et al. Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS. Plant Mol Biol Rep 26, 301–315 (2008). https://doi.org/10.1007/s11105-008-0043-6

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