Na⁺/K⁺exchange switches the catalytic apparatus of potassium-dependent plantL-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.

Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.)

“…The amino acids involved in anchoring the product in the active site and the catalytic switch are absolutely conserved in all the proteins in the alignment (Supplementary Figure S1), whereas those residues determining substrate specificity show some variability but with higher conservation among K + -dependent enzymes. According to Bejger et al (2014), K + -dependence is linked to the VMDKSPHS motif (shaded in gray and labeled as activation in Supplementary Figure S1), and it was proposed that the last serine residue is involved in the potassium-dependence mechanism. However, the PpASPG1 protein has an apolar valine residue in the last position of this motif instead of a polar serine residue.…”

“…As far as we know K + -dependent ASPG have been characterized in only four species: Pisum sativum (Sodek and Lea, 1993), A. thaliana (Bruneau et al, 2006; Gabriel et al, 2012), L. japonicus (Credali et al, 2011) and P. vulgaris (Bejger et al, 2014). Different findings have been published about the biochemical characteristics of plant ASPG.…”

“…The activation of plant ASPG has been addressed by various authors and described as an autoproteolytic maturation process in A. thaliana (Bruneau et al, 2006; Gabriel et al, 2012), L. luteus (Michalska et al, 2006), L. japonicus (Credali et al, 2011), and P. vulgaris (Bejger et al, 2014). In contrast, the ASPG from P. sylvestris expressed in E. coli did not show autocatalytic activity in vitro (Cañas et al, 2007).…”

“…Similar results were found in Lotus japonicus (Credali et al, 2011), but recent work on the Arabidopsis ASPG also reports a double activity asparaginase/isoaspartyl dipeptidase for the K + -dependent type (Gabriel et al, 2012). Crystallographic structure analysis of a K + -dependent ASPG from Phaseolus vulgaris showed a catalytic pocket big enough to host substrates larger than Asn, but it has been proposed that substrate specificity is probably determined by the nature of the amino acids in the vicinity of the substrate side chain(Bejger et al, 2014). The existence of these two subgroups within plants suggests a functional specialization for the isoforms.…”

“…When comparing protein sequences of ASPG isoforms and homologs in plants, high diversity is noticed in the region preceding the catalytic nucleophile, both in sequence as well as in length. This region, often referred to as a variable loop (VL), presents a lack of electron density in X-ray diffraction data of the protein crystals and therefore probably does not have a defined secondary structure (Michalska et al, 2006; Bejger et al, 2014). This has made it difficult to postulate a hypothesis about its function based on structural aspects.…”