title: Na⁺/K⁺ exchange switches the catalytic apparatus of potassium-dependent plant L-asparaginase.
abstract: Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K(+), Na(+) or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and β subunits, thus creating the mature heterotetramer or dimer of heterodimers (αβ)2. The αβ subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two β-sheets flanked on each side by a layer of helices. In addition to the `sodium loop' (here referred to as the `stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K(+) is coordinated) or OFF (when Na(+) is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.
authors.affiliation: Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bejger
authors.affiliation: Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Imiolczyk
authors.affiliation: Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.
authors.forename: Damien
authors.initials: D
authors.lastname: Clavel
authors.affiliation: Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Miroslaw
authors.initials: M
authors.lastname: Gilski
authors.affiliation: Agriculture and Agri-Food Canada, London, Ontario, Canada.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pajak
authors.affiliation: Agriculture and Agri-Food Canada, London, Ontario, Canada.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Marsolais
authors.affiliation: Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Jaskolski
mesh_terms: D001215:Asparaginase / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D003460:Crystallization; D017931:DNA Primers; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D012964:Sodium; D001215:Asparaginase; D011188:Potassium
keywords: K+-dependent enzyme; Ntn-hydrolase; Phaseolus vulgaris; amidohydrolase; isoaspartyl aminopeptidase; l-asparaginase; metal coordination