Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease

• Published in: Biochimica et biophysica acta 2011-12, Vol.1814 (12), p.1758-1768
• Main Authors: Mulinari, Fernanda, Becker-Ritt, Arlete Beatriz, Demartini, Diogo Ribeiro, Ligabue-Braun, Rodrigo, Stanisçuaski, Fernanda, Verli, Hugo, Fragoso, Rodrigo R., Schroeder, Evelyn Koeche, Carlini, Célia Regina, Grossi-de-Sá, Maria Fátima
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Sequence; ammonia; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - metabolism; Antifungal Agents - pharmacology; Antifungal protein; bacteria; Base Sequence; bicarbonates; Canavalia - chemistry; Canavalia - enzymology; Canavalia - genetics; Canavalia ensiformis; Cloning, Molecular; Comparative modeling; complementary DNA; diuresis; Escherichia coli; genes; Heterologous expression; hydrolysis; Insecticide; Isoenzymes - genetics; Isoenzymes - isolation & purification; Jackbean urease; Malpighian tubules; mass spectrometry; Microbial Sensitivity Tests; Molecular Sequence Data; Mutagenesis, Site-Directed; Ni metallocenter; nickel; peptides; Phylogeny; plant pathogenic fungi; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Proteins - pharmacology; protective effect; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Rhodnius prolixus; seed extracts; Sequence Homology, Nucleic Acid; urea; urease; Urease - genetics; Urease - isolation & purification; Urease - metabolism; Urease - pharmacology
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2011.07.022

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3′ and 5′ ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants. ► A cDNA encoding the isoform JBURE-IIb of Canavalia ensiformis urease was cloned. ► Comparative modeling and phylogenetic analysis of JBURE-IIb were performed. ► Proteomic analysis of C. ensiformis indicated that jbure-IIb is a functional gene. ► The full length jbure-IIb cDNA was cloned and expressed in Escherichia coli. ► Biological properties of JBURE-IIb point to a protective role of ureases in plants.