Abstract
Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.
Highlights
IntroductionUreases (urea amidohydrolase, EC 3.5.1.5) are nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and carbamate [1,2,3]
Ureases are nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and carbamate [1,2,3]
The gene coding for the Soyuretox 94-residue polypeptide (NCBI CAC43845.1) (Figure 1A) was cloned in the pET23a plasmid and expressed using E. coli BL21 (DE3) pLysS cells to reduce basal protein expression, obtaining a protein containing an His-tag at the C-terminus
Summary
Ureases (urea amidohydrolase, EC 3.5.1.5) are nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and carbamate [1,2,3] These proteins are widely spread in bacteria, plants, and fungi, but are not synthesized by animals [4,5]. Regardless of their origin and quaternary organization, ureases are homologous proteins that exhibit at least 55% identity at the amino acid sequence level. The insecticidal activity of C. ensiformis ureases depends, at least partially, on the hydrolysis of the protein and release of entomotoxic peptides upon the action of cathepsin-like digestive enzymes of the susceptible insects [7,10,11,12,13]. In order to rationalize the antifungal and entomotoxic effects of Jaburetox, NMR (nuclear magnetic resonance), CD (circular dichroism), and fluorescence spectroscopies were used to reveal the intrinsically disordered nature of this polypeptide and the modification in the polypeptide folding caused by interactions with lipid vesicles and planar lipid bilayers, as well as with yeast and cockroach membranes [21,22,23,24]
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