title: Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease.
abstract: 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.
authors: Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. fermulinari@pq.cnpq.br
authors: Fernanda
authors: F
authors: Mulinari
authors: Arlete Beatriz
authors: AB
authors: Becker-Ritt
authors: Diogo Ribeiro
authors: DR
authors: Demartini
authors: Rodrigo
authors: R
authors: Ligabue-Braun
authors: Fernanda
authors: F
authors: Stanisçuaski
authors: Hugo
authors: H
authors: Verli
authors: Rodrigo R
authors: RR
authors: Fragoso
authors: Evelyn Koeche
authors: EK
authors: Schroeder
authors: Célia Regina
authors: CR
authors: Carlini
authors: Maria Fátima
authors: MF
authors: Grossi-de-Sá
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D032449:Canavalia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D014510:Urease / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D014510:Urease