Structural basis of potassium activation in plant asparaginases

Abstract

l-asparaginases (EC 3.5.1.1) play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium-dependent (PvAspG1) and potassium-independent (PvAspG-T2) l-asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser-118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG-T2. Conversely, the PvAspG1-S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG-T2-I117S mutant had a similar half-maximal effective concentration (EC ) value for potassium activation, between 0.1 and 0.3 mm. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser-118 influences the ionization of specific functional groups in the enzyme-substrate complex. Together, the results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant l-asparaginases. ENZYME: l-Asparaginase (EC 3.5.1.1).

Constitutive expression of Asparaginase in Gossypium hirsutum triggers insecticidal activity against Bemisia tabaci

“…Before genetic transformation, these analyses were performed to ensure the proper conformation and folding of targeted protein. From homology modelling we can infers that the ZmASN protein has close resemblance to the human threonine aspartase (2a8i) while the presence of conserved residues of PDB ID:2gez (potassium-independent plant asparaginase) in the active site of the ZmASN protein confirmed that it is the K + -independent asparaginase with Thr267 as its nucleophile, which is in accordance with the study by 2,35 where Thr293 was the nucleophile. The ZmASN possess K + -independent L-asparaginase activity as determined by in silico analysis whereas due to its structural similarity to 2a8i (human taspase1) it may also possess isoaspartyl aminopeptidase activity.…”

Structural and biophysical studies of new L-asparaginase variants: lessons from random mutagenesis of the prototypic Escherichia coli Ntn-amidohydrolase

“…The random mutagenesis of the EcAIII protein presented in this work is the first attempt to investigate how well the prototypic Ntn-hydrolase can tolerate multiple simultaneous substitutions in the active site. Some single site-directed mutagenesis experiments have been performed previously for similar enzymes to elucidate the catalytic and autoproteolytic mechanisms (Nomme et al, 2014;Michalska et al, 2008;Ajewole et al, 2018). Our sequencing analysis showed that the frequencies of residues at selected mutation sites are not uniform, but this is a typical result of local randomization with the use of degenerate codons (Tang et al, 2012).…”

Structural and biophysical aspects of L-asparaginases: a growing family with amazing diversity

“…Ser is replaced by isoleucine, the enzyme loses its potassium dependence. Conversely, introduction of serine at the corresponding position in the potassium-independent enzyme from P. vulgaris endowed it with potassium sensitivity (Ajewole et al, 2018). Interestingly, it was also discovered that in the crystal structure of the D90E active-site mutant of the Class 1 EcAII enzyme there is a conserved zinc-binding site formed by Asp100, His197 and Asp200 (Borek et al, 2014; Supplementary Fig.…”