title: Role of lysine and acidic amino acid residues on the insecticidal activity of Jackbean urease.
abstract: Canavalia ensiformis has three isoforms of urease: Jackbean urease (JBU), Jackbean urease II and canatoxin. These isoforms present several biological activities, independent from the enzymatic property, such as entomotoxicity and antifungal properties. The entomotoxic activity is a property of the whole protein, as well as of a 10 kDa peptide released by insect digestive enzymes. Here we have used chemical modification to observe the influence of lysines and acidic residues on JBU enzymatic and insecticidal activities. Chemical modification of lysine residues was performed with dimethylamine-borane complex and formaldehyde, and acidic residues were modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and ethylenediamine. Derivatized ureases, called JBU-Lys (lysine-modified) and JBU-Ac (acidic residues-modified), were assayed for their biochemical and insecticidal properties. Neither modification altered significantly the kinetic parameters analyzed, indicating that no residue critical for the enzyme activity was affected and that the modifications did not incur in any significant structural alteration. On the other hand, both modifications reduced the toxic activity of the native protein fed to Dysdercus peruvianus. The changes observed in the entomotoxic property of the derivatized proteins reflect alterations in different steps of JBU's toxicity towards insects. JBU-Ac is not susceptible to hydrolysis by insect digestive enzymes, hence impairing the release of toxic peptide(s), while JBU-Lys is processed as the native protein. On the other hand, the antidiuretic effect of JBU on Rhodnius prolixus is altered in JBU-Lys, but not in JBU-Ac. Altogether, these data emphasize the role of lysine and acidic residues on the insecticidal properties of ureases.
authors.affiliation: Department of Biophysics, Universidade Federal do Rio Grande do Sul, Porto Alegre CEP 91501-970, RS, Brazil. rafael.guerra@ufrgs.br
authors.forename: Rafael
authors.initials: R
authors.lastname: Real-Guerra
authors.forename: Célia Regina
authors.initials: CR
authors.lastname: Carlini
authors.forename: Fernanda
authors.initials: F
authors.lastname: Stanisçuaski
mesh_terms: D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000737:chemistry*; D000818:Animals; D050034:Antidiuretic Agents / Q000494:pharmacology; D032449:Canavalia / Q000201:enzymology*; D006868:Hydrolysis; D007306:Insecticides / Q000494:pharmacology*; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000494:pharmacology; D012239:Rhodnius; D014118:Toxins, Biological / Q000737:chemistry / Q000494:pharmacology; D014510:Urease / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D024342:Amino Acids, Acidic; D050034:Antidiuretic Agents; D007306:Insecticides; D010940:Plant Proteins; D020033:Protein Isoforms; D014118:Toxins, Biological; C032815:canatoxin; D014510:Urease; D008239:Lysine
keywords: CNTX; Canavalia ensiformis; Chemical modification; Dysdercus peruvianus; Insecticide; JBUre-I; JBUre-II; Rhodnius prolixus; Urease; canatoxin; jackbean urease; jackbean urease isoform II